Novel FGF homologs

ABSTRACT

The present invention relates to methods of using zFGF5 compositions to proliferate chondrocytes and their progenitors, and to induce deposition of cartilage. zFGF5 compositions are disclosed for treating disorders associated with chondrocytes, such as cartilage injuries and defects. In addition, methods for treating neurological disorders, such as stroke, are disclosed, and methods for using zFGF5 compositions to stimulate growth of cells associated with neurological injury and disease are disclosed.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser.No.09/634,318 which is related to U.S. patent application Ser. No.09/574,750, filed on May 18, 2000; U.S. patent application Ser. No.09/229,947, filed on Jan. 13, 1999; and U.S. Pat. No. 5,989,866, filedon Oct. 16, 1997; and Provisional Application 60/028,646, filed on Oct.16, 1996; for which claims of benefit are made under 35 U.S.C. §119(e)(1) and 35 U.S.C. § 120, and are incorporated herein by referencein their entirety.

BACKGROUND OF THE INVENTION

[0002] The fibroblast growth factor (FGF) family consists of at leasteighteen distinct members (Basilico et al., Adv. Cancer Res. 59:115-165,1992 and Fernig et al., Prog. Growth Factor Res. 5(4):353-377, 1994)which generally act as mitogens for a broad spectrum of cell types. Forexample, basic FGF (also known as FGF-2) is mitogenic in vitro forendothelial cells, vascular smooth muscle cells, fibroblasts, andgenerally for cells of mesoderm or neuroectoderm origin, includingcardiac and skeletal myocytes (Gospodarowicz et al., J. Cell. Biol.70:395-405, 1976; Gospodarowicz et al., J. Cell. Biol. 89:568-578, 1981and Kardami, J. Mol. Cell. Biochem. 92:124-134, 1990). In vivo, bFGF hasbeen shown to play a role in avian cardiac development (Sugi et al.,Dev. Biol. 168:567-574, 1995 and Mima et al., Proc. Natl. Acad. Sci.92:467-471, 1995), and to induce coronary collateral development in dogs(Lazarous et al., Circulation 94:1074-1082, 1996). In addition,non-mitogenic activities have been demonstrated for various members ofthe FGF family. Non-proliferative activities associated with acidicand/or basic FGF include: increased endothelial release of tissueplasminogen activator, stimulation of extracellular matrix synthesis,chemotaxis for endothelial cells, induced expression of fetalcontractile genes in cardiomyocytes (Parker et al., J. Clin. Invest.85:507-514, 1990), and enhanced pituitary hormonal responsiveness (Bairdet al., J. Cellular Physiol. 5:101-106, 1987.)

[0003] Several members of the FGF family do not have a signal sequence(aFGF, bFGF and possibly FGF-9) and thus would not be expected to besecreted. In addition, several of the FGF family members have theability to migrate to the cell nucleus (Friesel et al., FASEB 9:919-925,1995). All the members of the FGF family bind heparin based onstructural similarities. Structural homology crosses species, suggestinga conservation of their structure/function relationship (Ornitz et al.,J. Biol. Chem. 271(25):15292-15297, 1996.)

[0004] There are four known cellular FGF receptor genes (FGFRs), andthey are all tyrosine kinases. In general, the FGF family members bindto all of the known FGFRs, however, specific FGFs bind to specificreceptors with higher degrees of affinity. Another means for specificitywithin the FGF family is the spatial and temporal expression of theligands and their receptors during embryogenesis. Evidence suggests thatthe FGFs most likely act only in autocrine and/or paracrine manner, dueto their heparin binding affinity, which limits their diffusion from thesite of release (Flaumenhaft et al., J. Cell. Biol. 111(4):1651-1659,1990.) Basic FGF lacks a signal sequence, and is therefore restricted toparacrine or autocrine modes of action. It has been postulated thatbasic FGF is stored intracellularly and released upon tissue damage.Basic FGF has been shown to have two receptor binding regions that aredistinct from the heparin binding site (Abraham et al.,. EMBO J.5(10):2523-2528, 1986.)

[0005] It has been shown that FGFR-3 plays a role in bone growth. Micemade homozygous null for the FGFR-3 (−/−) resulted in postnatal skeletalabnormalities (Colvin et al.,Nature Genet. 12:309-397, 1996 and Deng etal., Cell 84:911-921, 1996). The mutant phenotype suggests that innormal mice, FGFR-3 plays a role in regulation of chondrocyte celldivision in the growth plate region of the bone (Goldfarb, Cytokine andGrowth Factor Rev. 7(4):311-325, 1996). The ligand for the FGFR-3 in thebone growth plate has not been identified.

[0006] Although four FGFRs have been identified, all of which have beenshown to have functional splice variants, the possibility that novel FGFreceptors exist is quite likely. For example, no receptor has beenidentified for the FGF-8a isoform (MacArthur et al., J. Virol.69(4):2501-2507, 1995.).

[0007] FGF-8 is a member of the FGF family that was originally isolatedfrom mammary carcinoma cells as an androgen-inducible mitogen. It hasbeen mapped to human chromosome 10q25-q26 (White et al., Genomics30:109-11, 1995.) FGF-8 is involved in embryonic limb development (Vogelet al., Development 122:1737-1750, 1996 and Tanaka et al., CurrentBiology 5(6):594-597, 1995.) Expression of FGF-8 during embryogenesis incardiac, urogenital and neural tissue indicates that it may play a rolein development of these tissues (Crossley et al., Development121:439-451, 1995.) There is some evidence that acrocephalosyndactylia,a congenital condition marked by peaked head and webbed fingers andtoes, is associated with FGF-8 point mutations (White et al., 1995,ibid.)

[0008] FGF-8 has five exons, in contrast to the other known FGFs, whichhave only three exons. The first three exons of FGF-8 correspond to thefirst exon of the other FGFs (MacArthur et al., Development121:3603-3613, 1995.) The human gene for FGF-8 codes for four isoformswhich differ in their N-terminal regions: FGF isoforms a, b, e, and f;in contrast to the murine gene which gives rise to eight FGF-8 isoforms(Crossley et al., 1995, ibid.) Human FGF-8a and FGF-8b have 100%homology to the murine proteins, and FGF-8e and FGF-8f proteins are 98%homologous between human and mouse (Gemel et al., Genomics 35:253-257,1996.)

[0009] Heart disease is the major cause of death in the United States,accounting for up to 30% of all deaths. Myocardial infarction (MI)accounts for 750,000 hospital admissions per year in the U.S., with morethan 5 million people diagnosed with coronary disease. Risk factors forMI include diabetes mellitus, hypertension, truncal obesity, smoking,high levels of low density lipoprotein in the plasma or geneticpredisposition.

[0010] Cardiac hyperplasia is an increase in cardiac myocyteproliferation, and has been demonstrated to occur with normal aging inthe human and rat (Olivetti et al., J. Am. Coll. Cardiol. 24(1):140-9,1994 and Anversa et al., Circ. Res. 67:871-885, 1990), and incatecholamine-induced cardiomyopathy in rats (Deisher et al., Am. J.Cardiovasc. Pathol. 5(1):79-88, 1994.) Whether the increase in myocytesoriginate with some progenitor cell, or are a result of proliferation ofa more terminally differentiated cell type, remains controversial.

[0011] However, because infarction and other causes of myocardialnecrosis appear to be irreparable, it appears that the normal mechanismsof cardiac hyperplasia cannot compensate for extensive myocyte death,and there remains a need for exogenous factors that promote hyperplasiaand ultimately result in renewal of the heart's ability to function.

[0012] Stroke is caused by either cerebral thrombosis, embolism, orsubarachnoid or cerebral hemorrhage, and results in ischemia inapproximately 80% of occurrences. Stroke is a major health problemdisabling over three million people in the United States, with 550,0000Americans suffering stroke each year, of which 150,000 of those affectedwill die. The current treatments to prevent tissue damage resulting fromstroke are very limited and require administration within an hour ofonset of the stroke. While there are more drugs available to try toprevent reoccurrence of stroke, they are not without some seriousdrawbacks, including the development of intracranial hemorrhaging,gastrointestinal bleeding and neutropenia. Therefore, any therapeuticsthat promote angiogenesis, promote neurite outgrowth, or survival ofneurons in necrotic areas of the central nervous system with somespecificity will be valuable. The molecules of the present inventionhave been shown to promote growth in specific tissues, includingneuronal tissue.

[0013] Bone remodeling is the dynamic process by which tissue mass andskeletal architecture are maintained. The process is a balance betweenbone resorption and bone formation, with two cell types thought to bethe major players. These cells are the osteoblast and osteoclast.Osteoblasts synthesize and deposit matrix to become new bone. Theactivities of osteoblasts and osteoclasts are regulated by many factors,systemic and local, including growth factors.

[0014] While the interaction between local and systemic factors has notbeen completely elucidated, there does appear to be consensus thatgrowth factors play a key role in the regulation of both normal skeletalremodeling and fracture repair.. Some of the growth factors that havebeen identified in bone include: IGF-I, IGF-II, TGF-β₁, TGF-β₂, bFGF,aFGF, PDGF and the family of bone morphogenic proteins (Baylink et al.,J. Bone Mineral Res. 8 (Supp. 2):S565-S572, 1993).

[0015] When bone resorption exceeds bone formation, a net loss in boneresults, and the propensity for fractures is increased. Decreased boneformation is associated with aging and certain pathological states. Inthe U.S. alone, there are approximately 1.5 million fractures annuallythat are attributed to osteoporosis. The impact of these fractures onthe quality of the patient's life is immense. Associated costs to thehealth care system in the U.S. are estimated to be $5-$10 billionannually, excluding long-term care costs.

[0016] Other therapeutic applications for growth factors influencingbone remodeling include, for example, the treatment of injuries whichrequire the proliferation of osteoblasts to heal, such as fractures, aswell as stimulation of mesenchymal cell proliferation and the synthesisof intramembraneous bone which have been indicated as aspects offracture repair (Joyce et al. 36th Annual Meeting, Orthopaedic ResearchSociety, Feb. 5-8, 1990. New Orleans, La.).

[0017] Replacement of damaged articular cartilage caused either byinjury or defect is a major challenge for physicians, and availabletreatments are considered unpredictable and effective for only a limitedtime. Therefore, the majority of younger patients either do not seektreatment or are counseled to postpone treatment for long as possible.When treatment is required, the standard procedure is a total jointreplacement or penetration of the subchondral bone to stimulatefibrocartilage deposition by chondrocytes. While deposition offibrocartilage is not a functional equivalent of articular cartilage, itis at the present the best available treatment because there has beenlittle success in replacing articular cartilage. Two approaches tostimulating deposition of articular cartilage that are beinginvestigated are: stimulating chondrocyte activity in vivo and ex vivoexpansion of chondrocytes and their progenitors for transplantation(Jackson et al., Arthroscopy: The J. of Arthroscopic and Related Surg.12:732-738, 1996). In addition, regeneration or repair of elasticcartilage is valuable for treating injuries and defects to ear and nose.Any growth factor with specificity for chondrocytes lineage cells thatstimulates those cells to growth, differentiate or induce cartilageproduction would be valuable for maintaining, repairing or replacingarticular cartilage.

[0018] The present invention provides such polypeptides for these andother uses that should be apparent to those skilled in the art from theteachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 and FIG. 2 illustrate a multiple alignment of humanfibroblast growth factor homologous factor 1 (FHF-1; SEQ ID NO: 21),human myocyte-activating factor (FGF-10; SEQ ID NO: 22), humanfibroblast growth factor homologous factor 4 (FHF-4; SEQ ID NO: 23),human fibroblast growth factor homologous factor 2 (FHF-2; SEQ ID NO:24), human fibroblast growth factor homologous factor 3 (FHF-3; SEQ IDNO: 25), human FGF-4 (SEQ ID NO: 26), human FGF-6 (SEQ ID NO: 27), humanFGF-2 (basic; SEQ ID NO: 28), human FGF-1 (acidic; SEQ ID NO: 29), humankeratinocyte growth factor 2 (KGF-2; SEQ ID NO: 30), human keratinocytegrowth factor precursor (FGF-7; SEQ ID NO: 31), human zFGF5 (SEQ ID NO:2), human FGF-8 (SEQ ID NO: 32) human FGF-5 (SEQ ID NO: 33), human FGF-9(SEQ ID NO: 34), and human FGF-3 (SEQ ID NO: 35). “*” designatesconserved amino acids; “:” designates conserved amino acidsubstitutions; and “.” designates less stringently conserved amino acidsubstitutions.

[0020]FIG. 3 is an inter-family similarity matrix illustrating thepercent identity between: (1) human FGF-5 (SEQ ID NO: 33), (2) humanFGF-6 (SEQ ID NO: 27), (3) human FGF-7 (SEQ ID NO: 31), (4) human FGF-8(SEQ ID NO: 32), (5) human FGF-9 (SEQ ID NO: 34), (6) human zFGF5 (SEQID NO: 2), (7) human FGF-10 (SEQ ID NO: 22), (8) human FGF-1 (SEQ ID NO:29), (9) human FHF-1 (SEQ ID NO: 21), (10) human FGF-2 (SEQ ID NO: 28),(II) human FHF-2 (SEQ ID NO: 24), (12) human FHF-4 (SEQ ID NO: 23), (13)human FGF-3 (SEQ ID NO: 35), (14) human KGF-2 (SEQ ID NO: 30), (15)human FHF-3 (SEQ ID NO: 25), and (16) human FGF-4 (SEQ ID NO: 26).

[0021]FIG. 4 is a multiple alignment of the amino acid sequences formature human zFGF5 and mouse zFGF5 (SEQ ID NOS: 2 and 39, respectively).

DETAILED DESCRIPTION OF THE INVENTION

[0022] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms:

[0023] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, FlagTM peptide (Hopp et al., Biotechnology 6:1204-10,1988), streptavidin binding peptide, or other antigenic epitope orbinding domain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0024] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

[0025] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0026] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M-⁻¹.

[0027] The term “complements of a polynucleotide molecule” is apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

[0028] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0029] The term “expression vector” is used to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

[0030] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985).

[0031] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

[0032] The term “operably linked”, when referring to DNA segments,indicates that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiates inthe promoter and proceeds through the coding segment to the terminator.

[0033] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0034] A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (¢nt∞), or kilobases(¢kb∞). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

[0035] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0036] The term “promoter” is used herein for its art-recognized meaningto denote a portion of a gene containing DNA sequences that provide forthe binding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

[0037] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0038] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

[0039] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger polypeptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0040] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0041] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0042] All references cited herein are incorporated by reference intheir entirety.

[0043] The present invention is based in part upon the discovery of anovel DNA sequence that encodes a fibroblast growth factor (FGF) homologpolypeptide having homology to FGF-8 and FGF-17 (Hoshikawa et.al.,Biochem. Biophys. Res. Comm. 244:187-191, 1998). Analysis of the tissuedistribution of the human mRNA corresponding to this novel DNA showedthat expression was highest in fetal heart tissue and adult hearttissue, followed by apparent but decreased expression levels in fetallung, skeletal muscle, smooth muscle tissues such as small intestine,colon and trachea. The FGF homolog polypeptide has been designatedzFGF5.

[0044] Tissue distribution in murine species does not appear tocompletely correspond with expression in human tissues. Northernanalysis of mouse tissues revealed that expression of mouse zFGF5 ishighest in spleen and day 17 embryo, followed by relatively lowerexpression in heart, lung, kidney and testis. Mouse heart tissueanalysis found expression highest in day 16 fetal heart tissue, withexpression in adult heart present in most mouse strains. It also appearsthat there may be variability within murine expression levels andtissues (Hu et al., Mol. Cell. Biol. 18:6063-6074, 1998; Ohbayashi etal., J. Biol. Chem. 273:18161-18164, 1998 and Maruoka et al., Mech.Develop. 74:175-175, 1998).

[0045] The nucleotide sequence of the zFGF5 cDNA is described in SEQ IDNO. 1, and its deduced amino acid sequence is described in SEQ ID NO. 2.When amino acid residue 28 (Glu) to amino acid residue 181 (Gln) of SEQID NO: 2 is compared to the corresponding region of FGF-8 (See FIGS. 1and 2) the aligned and deduced amino acid sequence has approximately 56%identity. FGF-17 (Hoshiwara et al., Biochem. Biophys. Res. Comm.244:187-191, 1998) has recently been identified, and has the highestdegree of homology to zFGF5. The region of highest identity is ˜66% overa 123 amino acid overlap which corresponds to the region of SEQ ID NO: 2from residue 55 (Tyr) to residue 177 (Arg).

[0046] The novel polypeptide encoded by the polynucleotide describedherein contains the CXFXE{6} Y motif present in all members of the FGFfamily. The CXFXE{6} Y motifs (SEQ ID NO: 36) are highly conserved. Aconsensus amino acid sequence of the CXFXEX{6} Y domain (SEQ ID NO: 36)includes human fibroblast growth factor homologous factor 1 (FHF-1;Smallwood et al., Proc. Natl. Acad. Sci. USA 93:9850-9857, 1996), humanmyocyte-activating factor (FGF-10; HSU76381, GENBANK identifier), humanfibroblast growth factor homologous factor 4 (FHF-4; Smallwood et al.,1996, ibid.), human fibroblast growth factor homologous factor 2 (FHF-2;Smallwood et al., 1996, ibid.), human fibroblast growth factorhomologous factor 3 (FHF-3; Smallwood et al., 1996, ibid.), human FGF-4(Basilico et al., Adv. Cancer Res. 59:115-165,1992), human FGF-6(Basilico et al., 1992, ibid.), human FGF-2 (basic; Basilico et al.,1992, ibid.), human FGF-1 (acidic; Basilico et al., 1992, ibid.), humankeratinocyte growth factor 2 (KGF-2; HSU67918 GENBANK identifier), humankeratinocyte growth factor precursor (FGF-7; Basilico et al., 1992,ibid.), human zFGF5, human FGF-8 (Gemel et al., Genomics 35:253-257,1996), human FGF-5 (Basilico et al., 1992, ibid.), human FGF-9 (Miyamotoet al., Mol. Cell. Biol. 13:4251-4259, 1993), human FGF-3 (Basilico etal., 1992, ibid.), and FGF-17 (Hoshiwara et al., 1998, ibid.).

[0047] Analysis of the cDNA encoding a zFGF5 polypeptide (SEQ ID NO: 1)revealed an open reading frame encoding 207 amino acids (SEQ ID NO: 2)comprising a mature polypeptide of 180 amino acids (residue 28 toresidue 207 of SEQ ID NO: 2). Multiple alignment of zFGF5 with otherknown FGFs revealed a block of high percent identity corresponding toamino acid residue 127 (Cys) to amino acid residue 138 (Tyr), of SEQ IDNO: 2 and is shown in FIG. 1. Several of the members of the FGF familydo not have signal sequences.

[0048] The mouse zFGF5 polynucleotide sequence as shown in SEQ ID NO: 38and corresponding amino acid sequence as shown in SEQ ID NO: 39 werefound to have a high degree of homology to that of the human ortholog.At the amino acid level, the mouse and human polypeptides areapproximately 98% identical, with three amino acid changes. The changesas shown in FIG. 4, correspond to a Val₂₆ in SEQ ID NO: 2 being Ala₂₆ inSEQ ID NO: 39 in the mouse polypeptide, Pro₁₈₃ in SEQ ID NO: 2 to Ala₁₈₃in SEQ ID NO: 39 and Ala₂₀₇ in SEQ ID NO: 2 to Gly₂₀₇ in SEQ ID NO: 39.As is noted previously, Ala₂₆ (mouse) and the corresponding Val₂₆(human) are in the secretory signal sequence, leaving only two aminoacid differences in the mature polypeptide. Based on the high identitybetween the mouse and human sequences, it is predicted that functionwill be equivalent as well. However, based on differences in tissuedistribution for the mouse and human expression, zFGF5 may have a widerorgan target distribution, and more diverse biological functions in themouse than in the human.

[0049] Members of the FGF farmily are characterized by heparin bindingdomains. A putative heparin-binding domain for zFGF5 has been identifiedin the region of amino acid residue 148 (Gly) to amino acid residue 169(Gln) of SEQ ID NO: 2 and SEQ ID NO: 39.

[0050] It is postulated that receptor-mediated signaling is initiatedupon binding of FGF ligand complexed with cell-surface heparin sulfateproteoglycans. Many FGF family members can be placed into one of tworelated families on the basis of their structures and functions. aFGFand bFGF consist of three exons separated by two introns of variablelength. FGF-8 consists of five exons, the first three of whichcorrespond to the first exon of aFGF and bFGF. All the known FGF familymembers are spliced to form single polypeptides.

[0051] Analysis of the ligand-receptor complex of zFGF-5 hasdemonstrated that zFGF-5 has specificity for the FGFR3α-IIIc and FGFR4receptors. Using cells that do not normally express any of the FGFreceptors but had been transfected to express FGFR1α-IIIb,-IIIc,FGFR2α-IIIb,-IIIc, or FGFR3α-IIIb, -IIIc, a high affinity cellproliferative response (EC50 ˜1-2 ng/ml) was seen with cells expressingthe FGFR3αIIIc. A lower affinity response (EC50 ˜10-20 ng/ml) ofactivation was observed in cells expressing FGFR2α-IIIc. Moreover, theactivation of the FGFR3αIIIc and FGFR2a-IIIc expressing cells by zFGF-5was completely dependent on exogenous heparin. Addition of zFGF-5 didnot enhance proliferation of FGFR1 nor any of the FGFR b splice variantexpressing cells. A comparison of FGF receptor specificity of FGF-1 andFGF-2 suggests that the receptor binding specificity of zFGF-5 isconsiderably more restricted than either FGF- 1 or -2 .

[0052] SEQ ID NO: 6 is a degenerate polynucleotide sequence thatencompasses all polynucleotides that could encode the zFGF5 polypeptideof SEQ ID NO: 2 (amino acids 1 or 28 to 207). Thus, zFGF5polypeptide-encoding polynucleotides ranging from nucleotide 1 or 82 tonucleotide 621 of SEQ ID NO: 6 are contemplated by the presentinvention. Also contemplated by the present invention are fragments andfusions as described above with respect to SEQ ID NO: 1, which areformed from analogous regions of SEQ ID NO: 6, wherein nucleotides 82 to621 of SEQ ID NO: 6 correspond to nucleotides 82 to 621 of SEQ ID NO: 1,for the encoding a mature zFGF5 molecule.

[0053] The symbols in SEQ ID NO: 6 are summarized in Table 1 below.TABLE 1 Nucleotide Resolutions Complement Resolutions A A T T C C G G GG C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G SC|G C|G A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T DA|G|T H A|C|T N A|C|G|T N A|C|G|T

[0054] The degenerate codons used in SEQ ID NO: 6, encompassing allpossible codons for a given amino acid, are set forth in Table 2 below.TABLE 2 Amino Degenerate Acid Letter Codons Codon Cys C TGC TGT TGY SerS AGC AGT TGA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCA CCCCCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn NAAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA GAG CAR His HCAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met MATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val VGTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGGTer . TAA TAG TGA TRR Asn/Asp B RAY Glu/Gln Z SAR Any X NNN Gap - ---

[0055] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may have some incorrect amino acids, but one of ordinary skillin the art can easily identify such erroneous sequences by reference tothe amino acid sequence of SEQ ID NO: 2.

[0056] The highly conserved amino acids in zFGF5 can be used as a toolto identify new family members. To identify new family members theconserved CXFXEX{6}Y motif (SEQ ID NO: 36) can be used. In anothermethod using polynucleotide probes and hybridization methods, RNAobtained from a variety of tissue sources can be used to generate cDNAlibraries and probe these libraries for new family members. Inparticular, reverse transcription-polymerase chain reaction (RT-PCR) canbe used to amplify sequences encoding highly degenerate DNA primersdesigned from the sequences corresponding to amino acid residue 127(Cys) to amino acid residue 138 (Tyr) of SEQ ID NO: 2.

[0057] Within certain embodiments of the invention the isolatedpolynucleotides will serve as a probe and hybridize to similar sizedregions of SEQ ID NO: 1 or a sequence complementary thereto, understringent conditions. In general, stringent conditions are selected tobe about 5° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typicalstringent conditions are those in which the salt concentration is atleast about 0.02 M at pH 7 and the temperature is at least about 60° C.

[0058] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for isolating DNA and RNA arewell known in the art. It is generally preferred to isolate RNA fromcardiac tissue, although DNA can also be prepared using RNA from othertissues or isolated as genomic DNA. Total RNA can be prepared usingguanidine HCl extraction followed by isolation by centrifugation in aCsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)+RNA is prepared from total RNA using the method of Aviv and Leder (Proc.Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA (cDNA) isprepared from poly(A)+ RNA using known methods. Polynucleotides encodingzFGF5 polypeptides are then identified and isolated by, for example,hybridization or PCR.

[0059] The present invention further provides counterpart polypeptidesand polynucleotides from other species (orthologs). Of particularinterest are zFGF5 polypeptides from other mammalian species, includingmurine, rat, porcine, ovine, bovine, canine, feline, equine and otherprimate proteins. Identification of variants of the human sequence areparticularly interesting because while eight variants of murine FGF-8have been identified, only four human variants are known. Human variantsor orthologs of the human proteins can be cloned using information andcompositions provided by the present invention in combination withconventional cloning techniques. For example, a cDNA can be cloned usingmRNA obtained from a tissue or cell type that expresses the protein.Suitable sources of mRNA can be identified by probing Northern blotswith probes designed from the sequences disclosed herein. A library isthen prepared from mRNA of a positive tissue or cell line. AzFGF5-encoding cDNA can then be isolated by a variety of methods, suchas by probing with a complete or partial human cDNA or with one or moresets of degenerate probes based on the disclosed sequences. A cDNA canalso be cloned using the polymerase chain reaction, or PCR (Mullis, U.S.Pat. No. 4,683,202), using primers designed from the sequences disclosedherein. Within an additional method, the cDNA library can be used totransform or transfect host cells, and expression of the CDNA ofinterest can be detected with an antibody to zFGF5. Similar techniquescan also be applied to the isolation of genomic clones.

[0060] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NO: 1 or SEQ ID NO: 38 and SEQ ID NO: 2 and SEQ IDNO: 39 represent a single allele of the human and mouse zFGF5 gene andpolypeptide, respectively, and that allelic variation and alternativesplicing are expected to occur. Allelic variants can be cloned byprobing cDNA or genomic libraries from different individuals accordingto standard procedures. Allelic variants of the DNA sequence shown inSEQ ID NO: 1 or SEQ ID NO: 38, including those containing silentmutations and those in which mutations result in amino acid sequencechanges, are within the scope of the present invention, as are proteinswhich are allelic variants of SEQ ID NO: 2 or SEQ ID NO: 39.

[0061] The present invention also provides isolated zFGF5 polypeptidesthat are substantially homologous to the polypeptides of SEQ ID NO: 2and their orthologs. The term “substantially homologous” is used hereinto denote polypeptides having 50% to 60%, with certain embodimentshaving at least 80%, sequence identity to the sequences shown in SEQ IDNO: 2 or their orthologs. In other embodiments, polypeptides may also beat least 90% identical to 95% or more identical to SEQ ID NO: 2 or itsorthologs. Percent sequence identity is determined by conventionalmethods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915-10919, 1992. Briefly, two amino acid sequences are aligned tooptimize the alignment scores using a gap opening penalty of 10, a gapextension penalty of 1, and the “blosum 62” scoring matrix of Henikoffand Henikoff (ibid.) as shown in Table 3 (amino acids are indicated bythe standard one-letter codes). The percent identity is then calculatedas:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}} \right. \\{{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}} \\\left. {{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

[0062] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0063] Substantially homologous proteins and polypeptides arecharacterized as having one or more amino acid substitutions, deletionsor additions. These changes are preferably of a minor nature, that isconservative amino acid substitutions (see Table 4) and othersubstitutions that do not significantly affect the folding or activityof the protein or polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or a small extension that facilitatespurification (an affinity tag), such as a poly-histidine tract, proteinA (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., MethodsEnzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson,Gene 67:31, 1988), maltose binding protein (Kellerman and Ferenci,Methods Enzymol. 90:459-463, 1982; Guan et al., Gene 67:21-30, 1987), orother antigenic epitope or binding domain. See, in general Ford et al.,Protein Expression and Purification 2: 95-107, 1991, which isincorporated herein by reference. DNAs encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.; New England Biolabs, Beverly, Mass.). TABLE 4Conservative amino acid substitutions Basic: arginine lysine histidineAcidic: glutamic acid aspartic acid Polar: glutamine asparagineHydrophobic: leucine isoleucine valine Aromatic: phenylalaninetryptophan tyrosine Small: glycine alanine serine threonine methionine

[0064] The proteins of the present invention can also comprise, inaddition to the 20 standard amino acids, non-naturally occurring aminoacid residues. Non-naturally occurring amino acids include, withoutlimitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl-glycine,allo-threonine, methylthreonine, hydroxyethyl-cysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutarmine, pipecolicacid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenyl-alanine, 4-fluorophenylalanine, 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline and a-methyl serine. Severalmethods are known in the art for incorporating non-naturally occurringamino acid residues into proteins. For example, an in vitro system canbe employed wherein nonsense mutations are suppressed using chemicallyaminoacylated suppressor tRNAs. Methods for synthesizing amino acids andarminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations are carried out in a cell freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Meth. Enzymol. .202:301, 1991; Chung et al., Science259:806-09, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-49, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-98, 1996). Within a third method, E. coli cells are culturedin the absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-76, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

[0065] Essential amino acids in the zFGF5 polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-1085, 1989). In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (e.g., receptor binding activity using ¹²⁵I-zFGF5(Moscatelli, J. Cell Physio. 131:123-130. 1987), activation of receptortyrosine kinase (Panek et al., J. Pharm. Exp. Therapeutics 286:569-577,1998 and Schafer et al., Anal. Biochem. 261:100-112, 1998), generationof cardiac myocytes or fibroblasts, or stimulation of bone formation) toidentify amino acid residues that are critical to the activity of themolecule. See also, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996.Sites of ligand-receptor interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306-312,1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al.,FEBS Lett. 309:59-64, 1992. The identities of essential amino acids canalso be inferred from analysis of homologies with related FGFs and areshown in FIGS. 1 and 2.

[0066] Analyses of the amino acid sequence of human and mouse zFGF5revealed a dibasic site at the C-terminus of the polypeptide (amino acidresidue 196-197 (Lys-Arg)). A C-terminally truncated polypeptidecomprising an amino acid sequence as shown in SEQ ID NO: 2, from aminoacid residue 28 (Glu) to amino acid residue 196 (Lys) was demonstratedto have biological activity. Dibasic amino acids, such as, Arg-X-X-Arg(wherein X is any amino acid residue; SEQ ID NO: 37), Arg-Arg orLys-Arg; are subject to cleavage by several enzymes, including, but notlimited to, thrombin and carboxypeptidases. Therefore, it is within thescope of the claims to make conservative changes at dibasic amino acidresidues, in particular the dibasic residues at amino acid residues 196and 197 (Lys and Arg, respectively) of SEQ ID NO: 2 or SEQ ID NO: 39.

[0067] Based on analyses of the FGF family a C-terminally truncatedmolecule that comprises amino acid residue 28 (Glu) to residue 175 (Met)of SEQ ID NO: 2 will be biologically active. An intramolecular disulfidebond is predicted to occur between amino acid residue 109 (Cys) andresidue 127 (Cys) of SEQ ID NO: 2 or SEQ ID NO: 39.

[0068] Based on homology alignments with FGF-1 and FGF-2 crystalstructures (Eriksson et al., Prot. Sci. 2:1274, 1993), secondarystructure predictions for beta strand structure of zFGF5 correlates toamino acid residues 56-59, 64-69, 73-76, 85-92, 96-102, 106-111,115-119, 128-134, 138-144, 149-155, and 173-177 of SEQ ID NO: 2 or SEQID NO: 39. Amino acids critical for zFGF5 binding to receptors can beidentified by site-directed mutagenesis of the entire zFGF5 polypeptide.More specifically, they can be identified using site-directedmutagenesis of amino acids in the zFGF5 polypeptide which correspond toamino acid residues in acidic FGF (FGF1) and basic FGF (FGF2) identifiedas critical for binding of these FGFs to their receptors (Blaber et al.,Biochem. 35:2086-2094, 1996). These amino acids include Tyr33, Arg53,Asn110, Tyr112, Lys119, Trp123, Leu149 and Met151 in human FGF2, andTyr30, Arg5O, Asn107, Tyr109, Lys116, Trp122, Leu148 and Leul150 inhuman FGF1, as shown in FIG. 1 and FIG 2. The corresponding amino acidsin zFGF5, as shown in FIG. 1 and FIG. 2, would be Tyr58, Gly77, Asn136,Tyr138, Lys145, Trp149, Met175 and Arg177. One skilled in the art willrecognize that other members, in whole or in part, of the FGF family mayhave structural or biochemical similarities to zFGF5, and be substitutedmaking such analyses. Such regions would be important for biologicalfunctions of the molecule.

[0069] An alignment based on homology of zFGF5 with FGF-17 revealed thehighest percent identity region consists of a 123 amino acid overlapfound between residue 55 (Tyr) and residue 177 (Arg) of SEQ ID NO: 2with ˜66% identity. When conservative amino acid changes are calculatedover the same region, the percent homology is ˜92%.

[0070] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

[0071] Mutagenesis methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides (e.g., cell proliferation) canbe recovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

[0072] Using the methods discussed above, one of ordinary skill in theart can identify and/or prepare a variety of polypeptides that aresubstantially homologous to residues 28 (Glu) to 175 (Met), residues 28(Glu) to 196 (Lys) or residues 28 (Glu) to 207 (Ala) of SEQ ID NO: 2,allelic variants thereof, or biologically active fragments thereof, andretain the proliferative properties of the wild-type protein. Suchpolypeptides may also include additional polypeptide segments asgenerally disclosed above.

[0073] The polypeptides of the present invention, including full-lengthproteins, fragments thereof and fusion proteins, can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly cultured cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are disclosed bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, andAusubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley and Sons, Inc., NY, 1987, which are incorporated herein byreference.

[0074] In general, a DNA sequence encoding a zFGF5 polypeptide of thepresent invention is operably linked to other genetic elements requiredfor its expression, generally including a transcription promoter andterminator within an expression vector. The vector will also commonlycontain one or more selectable markers and one or more origins ofreplication, although those skilled in the art will recognize thatwithin certain systems selectable markers may be provided on separatevectors, and replication of the exogenous DNA may be provided byintegration into the host cell genome. Selection of promoters,terminators, selectable markers, vectors and other elements is a matterof routine design within the level of ordinary skill in the art. Manysuch elements are described in the literature and are available throughcommercial suppliers.

[0075] To direct a zFGF5 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be the native sequence, or a chimeracomprising a signal sequence derived from another secreted protein(e.g., t-PA and α-pre-pro secretory leader) or synthesized de novo. Thesecretory signal sequence is joined to the zFGF5 DNA sequence in thecorrect reading frame. Secretory signal sequences are commonlypositioned 5′ to the DNA sequence encoding the polypeptide of interest,although certain signal sequences may be positioned elsewhere in the DNAsequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743;Holland et al., U.S. Pat. No. 5,143,830).

[0076] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). A preferred vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092), andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillernondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

[0077] The use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed in WIPO Publications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which are preferably linearizedprior to transformation. For polypeptide production in P. methanolica,it is preferred that the promoter and terminator in the plasmid be thatof a P. methanolica gene, such as a P. methanolica alcohol utilizationgene (AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DIIAS), formate dehydrogenase (FMD), andcatalase (CAT) genes. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolicais a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, it is preferred to use host cells in whichboth methanol utilization genes (AUG1 and AUG2) are deleted. Forproduction of secreted proteins, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (ω) of from1 to 40 milliseconds, most preferably about 20 milliseconds.

[0078] Other methods for transforming yeast cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). An alternative preferred vectorsystem for use in yeast is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Suitable promoters andterminators for use in yeast include those from glycolytic enzyme genes(see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S.Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092, which areincorporated herein by reference) and alcohol dehydrogenase genes. Seealso U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454, whichare incorporated herein by reference. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia guillermondii, and Candida maltosa are known in theart.. A particularly preferred system utilizes Pichia methanolica (see,PCT application WO 9717450). For alternative transformation systems,see, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349,which is incorporated herein by reference. Methods for transformingAcremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No.5,162,228, which is incorporated herein by reference. Methods fortransforming Neurospora are disclosed by Lambowitz, U.S. Pat. No.4,486,533, which is incorporated herein by reference.

[0079] Cultured mammalian cells are also preferred hosts within thepresent invention. Methods for introducing exogenous DNA into mammalianhost cells include calcium phosphate-mediated transfection (Wigler etal., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973),electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextranmediated transfection (Ausubel et al., eds., Current Protocols inMolecular Biology, John Wiley and Sons, Inc., NY, 1987), andliposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993;Ciccarone et al., Focus 15:80, 1993), which are incorporated herein byreference. The production of recombinant polypeptides in culturedmammalian cells is disclosed, for example, by Levinson et al., U.S. Pat.No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al.,U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134, which areincorporated herein by reference. Preferred cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen.Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-KI; ATCC No.CCL 61 or DG44) cell lines. Additional suitable cell lines are known inthe art and available from public depositories such as the American TypeCulture Collection, Rockville, Maryland. In general, strongtranscription promoters are preferred, such as promoters from SV-40 orcytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitablepromoters include those from metallothionein genes (U.S. Pat. Nos.4,579,821 and 4,601,978, which are incorporated herein by reference) andthe adenovirus major late promoter.

[0080] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

[0081] Other higher eukaryotic cells can also be used as hosts,including insect cells, plant cells and avian cells. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222; Bang et al., U.S. Pat. No.4,775,624; and WIPO publication WO 94/06463, which are incorporatedherein by reference. The use of Agrobacterium rhizogenes as a vector forexpressing genes in plant cells has been reviewed by Sinkar et al., J.Biosci. (Bangalore) 11:47-58, 1987.

[0082] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

[0083] Expressed recombinant zFGF5 polypeptides (or chimeric zFGF5polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitableanion exchange media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,Piscataway, NJ) being particularly preferred. Exemplary chromatographicmedia include those media derivatized with phenyl, butyl, or octylgroups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650(Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and thelike; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) andthe like. Suitable solid supports include glass beads, silica-basedresins, cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate. moieties. Examples of coupling chemistriesinclude cyanogen bromide activation, N-hydroxysuccinimide activation,epoxide activation, sulfhydryl activation, hydrazide activation, andcarboxyl and amino derivatives for carbodiimide coupling chemistries.These and other solid media are well known and widely used in the art,and are available from commercial suppliers. Methods for bindingreceptor polypeptides to support media are well known in the art.Selection of a particular method is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988.

[0084] The polypeptides of the present invention can also be isolated byexploitation of their heparin binding properties. For a review, see,Burgess et al., Ann. Rev. of Biochem. 58:575-606, 1989. Members of theFGF family can be purified to apparent homogeneity by heparin-Sepharoseaffinity chromatography (Gospodarowicz et al., Proc. Natl. Acad. Sci.81:6963-6967, 1984) and eluted using linear step gradients of NaCl (Ronet al., J. Biol. Chem. 268(4):2984-2988, 1993; Chromatography:Principles & Methods, pp. 77-80, Pharmacia LKB Biotechnology, Uppsala,Sweden, 1993; in “Immobilized Affinity Ligand Techniques”, Hermanson etal., eds., pp. 165-167, Academic Press, San Diego, 1992; Kjellen et al.,Ann. Rev. Biochem.Ann. Rev. Biochem. 60:443-474, 1991; and Ke et al.,Protein Expr. Purif. 3(6):497-507, 1992.)

[0085] Other purification methods include using immobilized metal ionadsorption (IMAC) chromatography to purify histidine-rich proteins.Briefly, a gel is first charged with divalent metal ions to form achelate (E. Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (Methodsin Enzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp.529-39). Alternatively, a fusionof the polypeptide of interest and an affinity tag (e.g., polyhistidine,maltose-binding protein, an immunoglobulin domain) may be constructed tofacilitate purification.

[0086] Protein refolding (and optionally reoxidation) procedures may beadvantageously used. It is preferred to purify the protein to >80%purity, more preferably to >90% purity, even more preferably >95%, andparticularly preferred is a pharmaceutically pure state, that is greaterthan 99.9% pure with respect to contaminating macromolecules,particularly other proteins and nucleic acids, and free of infectiousand pyrogenic agents. Preferably, a purified protein is substantiallyfree of other proteins, particularly other proteins of animal origin.zFGF5 polypeptides or fragments thereof may also be prepared throughchemical synthesis. zFGF5 polypeptides may be monomers or multimers;glycosylated or non-glycosylated; pegylated or non-pegylated; and may ormay not include an initial methionine amino acid residue.. PEGylation isone method commonly used that has been demonstrated to increase plasmahalf-life, increased solubility, and decreased antigenicity anddecreased immunogenicity (Nucci et al., Advanced Drug Delivery Reviews6:133-155, 1991 and Lu et al., Int. J. Peptide Protein Res. 43:127-138,1994). Several procedures have been reported to create and purifyPEGylated proteins (See, e.g., Abuchowski et al., J. Biol. Chem.252:3582-3586, 1977 and Becauchamp et al., Anal. Biochem. 131:25-33,1983.) PEGylation may be achieved by modification of carboxyl amino acidresidues of a polypeptide or protein. In particular acid amino acidresidues (e.g. glutamic and aspartic acids) and amino acids at thecarboxyl-terminus are amenable to PEGylation (Zalipsky, BioconjugateChem. 6:150-165, 1995).

[0087] The activity of molecules of the present invention can bemeasured using a variety of assays that, for example, measure neogenesisor hyperplasia (i.e., proliferation) of cardiac cells based on thetissue specificity in adult heart. Additional activities likelyassociated with the polypeptides of the present invention includeproliferation of endothelial cells, cardiomyocytes, chondrocytes,fibroblasts, skeletal myocytes directly or indirectly through othergrowth factors; action as a chemotaxic factor for endothelial cells,fibroblasts and/or phagocytic cells; osteogenic factor; and factor forexpanding mesenchymal stem cell and precursor populations.

[0088] Proliferation can be measured using cultured cardiac cells or invivo by administering molecules of the claimed invention to theappropriate animal model. Generally, proliferative effects are seen asan increase in cell number and therefore, may include inhibition ofapoptosis, as well as mitogenesis. Cultured cells include cardiacfibroblasts, cardiac myocytes, skeletal myocytes, chondrocytes, humanumbilical endothelial vein cells from primary cultures. Established celllines include: NIH 3T3 fibroblast (ATCC No. CRL-1658), CHH-1 chum heartcells (ATCC No. CRL-1680), H9c2 rat heart myoblasts (ATCC No. CRL-1446),Shionogi mammary carcinoma cells (Tanaka et al., Proc. Natl . Acad. Sci.89:8928-8932, 1992) and LNCap.FGC adenocarcinoma cells (ATCC No.CRL-1740.) Assays measuring cell proliferation are well known in theart. For example, assays measuring proliferation include such assays aschemosensitivity to neutral red dye (Cavanaugh et al., InvestigationalNew Drugs 8:347-354, 1990, incorporated herein by reference),incorporation of radiolabelled nucleotides (Cook et al., AnalyticalBiochem. 179:1-7, 1989, incorporated herein by reference), incorporationof 5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells(Porstmann et al., J. Immunol. Methods 82:169-179, 1985, incorporatedherein by reference), and use of tetrazolium salts (Mosmann, J. Immunol.Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988;Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., CancerRes. 48:4827-4833, 1988; all incorporated herein by reference).

[0089] Differentiation is a progressive and dynamic process, beginningwith pluripotent stem cells and ending with terminally differentiatedcells. Pluripotent stem cells that can regenerate without commitment toa lineage express a set of differentiation markers that are lost whencommitment to a cell lineage is made. Progenitor cells express a set ofdifferentiation markers that may or may not continue to be expressed asthe cells progress down the cell lineage pathway toward maturation.Differentiation markers that are expressed exclusively by mature cellsare usually functional properties such as cell products, enzymes toproduce cell products and receptors. The stage of a cell population'sdifferentiation is monitored by identification of markers present in thecell population. Myocytes, osteoblasts, adipocytes, chondrocytes,fibroblasts and reticular cells are believed to originate from a commonmesenchymal stem cell (Owen et al., Ciba Fdn. Symp. 136:42-46, 1988).Markers for mesenchymal stem cells have not been well identified (Owenet al., J. of Cell Sci. 87:731-738, 1987), so identification is usuallymade at the progenitor and mature cell stages. The existence of earlystage cardiac myocyte progenitor cells (often referred to as cardiacmyocyte stem cells) has been speculated, but not demonstrated, in adultcardiac tissue. However, recent evidence confirms the presence ofmyocyte proliferation in end-stage cardiac failure in humans (Kajsturaet al., Proc. Natl. Assoc. Science, 95:8801-8805, 1998). The novelpolypeptides of the present invention are useful to isolate mesenchymalstem cells and cardiac myocyte progenitor cells, both in vivo and exvivo.

[0090] There is evidence to suggest that factors that stimulate specificcell types down a pathway towards terminal differentiation ordedifferentiation, affects the entire cell population originating from acommon precursor or stem cell. Thus, the present invention includesstimulating inhibition or proliferation of myocytes, smooth musclecells, osteoblasts, adipocytes, chondrocytes and endothelial cells.Molecules of the present invention may, while stimulating proliferationor differentiation of cardiac myocytes, inhibit proliferation ordifferentiation of adipocytes, by virtue of the affect on their commonprecursor/stem cells. Thus molecules of the present invention, have usein inhibiting osteosarcomas, chondro-sarcomas, atherosclerosis,restenosis, osteoporosis and obesity.

[0091] Assays measuring differentiation include, for example, measuringcell-surface markers associated with stage-specific expression of atissue, enzymatic activity, functional activity or morphological changes(Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994;Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; allincorporated herein by reference).

[0092] In vivo assays for evaluating cardiac neogenesis or hyperplasiainclude treating neonatal and mature rats with the molecules of thepresent invention. The animals cardiac function is measured as heartrate, blood pressure, and cardiac output to determine left ventricularfunction. Post-mortem methods for assessing cardiac improvement include:increased cardiac weight, nuclei/cytoplasmic volume, staining of cardiachistology sections to determine proliferating cell nuclear antigen(PCNA) vs. cytoplasmic actin levels (Quaini et al., Circulation Res.75:1050-1063, 1994 and Reiss et al., Proc. Nati. Acad. Sci.93:8630-8635, 1996.)

[0093] In vivo assays for measuring changes in bone formation ratesinclude performing bone histology (see, Recker, R., eds. BoneHistomorphometry: Techniques and Interpretation. Boca Raton: CRC Press,Inc., 1983) and quantitative computed tomography (QCT; Ferretti,J. Bone17:353S-364S, 1995; Orphanoludakis et al., Investig. Radiol.14:122-130,, 1979 and Durand et al., Medical Physics 19:569-573, 1992).An ex vivo assay for measuring changes in bone formation would be, forexample, a calavarial assay (Gowen et al., J. Immunol. 136:2478-2482,1986).

[0094] With regard to modulating energy balance, particularly as itrelates to adipocyte metabolism, proliferation and differentiation,zFGF5 polypeptides modulate effects on metabolic reactions. Suchmetabolic reactions include adipogenesis, gluconeogenesis,glycogenolysis, lipogenesis, glucose uptake, protein synthesis,thermogenesis, oxygen utilization and the like. Among other methodsknown in the art or described herein, mammalian energy balance may beevaluated by monitoring one or more of the aforementioned metabolicfunctions. These metabolic functions are monitored by techniques (assaysor animal models) known to one of ordinary skill in the art, as is morefully set forth below. For example, the glucoregulatory effects ofinsulin are predominantly exerted in the liver, skeletal muscle andadipose tissue. In skeletal muscle and adipose tissue, insulin acts tostimulate the uptake, storage and utilization of glucose.

[0095] Art-recognized methods exist for monitoring all of the metabolicfunctions recited above. Thus, one of ordinary skill in the art is ableto evaluate zFGF5 polypeptides, fragments, fusion proteins, antibodies,agonists and antagonists for metabolic modulating functions. Exemplarymodulating techniques are set forth below.

[0096] Insulin-stimulated lipogenesis, for example, may be monitored bymeasuring the incorporation of ¹⁴C-acetate into triglyceride (Mackall etal. J. Biol. Chem. 251:6462-6464, 1976) or triglyceride accumulation(Kletzien et al., Mol. Pharmacol. 41:393-398, 1992).

[0097] zFGF5-stimulated uptake may be evaluated, for example, in anassay for insulin-stimulated glucose transport. Primary adipocytes orNIH 3T3 LI cells (ATCC No. CCL-92.1) are placed in DMEM containing 1 g/lglucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mM glutamine. After two tofive hours of culture, the medium is replaced with fresh, glucose-freeDMEM containing 0.5 or 1.0% BSA, 20 mM Hepes, 1 mM pyruvate, and 2 mMglutamine. Appropriate concentrations of zFGF5, insulin or IGF-1, or adilution series of the test substance, are added, and the cells areincubated for 20-30 minutes. ³H or ¹⁴C-labeled deoxyglucose is added to≈50 μM final concentration, and the cells are incubated forapproximately 10-30 minutes. The cells are then quickly rinsed with coldbuffer (e.g. PBS), then lysed with a suitable lysing agent (e.g. 1% SDSor 1 N NaOH). The cell lysate is then evaluated by counting in ascintillation counter. Cell-associated radioactivity is taken as ameasure of glucose transport after subtracting non-specific binding asdetermined by incubating cells in the presence of cytochalasin b, aninhibitor of glucose transport. Other methods include those describedby, for example, Manchester et al., Am. J. Physiol. 266 (Endocrinol.Metab. 29):E326-E333, 1994 (insulin-stimulated glucose transport).

[0098] Protein synthesis may be evaluated, for example, by comparingprecipitation of ³⁵S-methionine-labeled proteins following incubation ofthe test cells with ³⁵S-methionine and ³⁵S-methionine and a putativemodulator of protein synthesis.

[0099] Thermogenesis may be evaluated as described by B. Stanley in TheBiology of Neuropeptide Y and Related Peptides, W. Colmers and C.Wahlestedt (eds.), Humana Press, Ottawa, 1993, pp. 457-509; C.Billington et al., Am. J. Physiol. 260:R321, 1991; N. Zarjevski et al.,Endocrinology 133:1753, 1993; C. Billington et al., Am. J. Physiol.266:R1765, 1994; Heller et al., Am. J. Physiol. 252(4 Pt 2): R661-7,1987; and Heller et al., Am. J. Physiol. 245(3): R321-8, 1983. Also,metabolic rate, which may be measured by a variety of techniques, is anindirect measurement of thermogenesis.

[0100] Oxygen utilization may be evaluated as described by Heller etal., Pflugers Arch. 369(1): 55-9, 1977. This method also involved ananalysis of hypothalmic temperature and metabolic heat production.Oxygen utilization and thermoregulation have also been evaluated inhumans as described by Haskell et al., J. Appl. Physiol. 51(4): 948-54,1981.

[0101] zFGF5 polypeptides can also be used to prepare antibodies thatspecifically bind to zFGF5 epitopes, peptides or polypeptides. Methodsfor preparing polyclonal and monoclonal antibodies are well known in theart (see, for example, Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., 1989; and Hurrell, J.G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla., 1982, which areincorporated herein by reference). As would be evident to one ofordinary skill in the art, polyclonal antibodies can be generated from avariety of warm-blooded animals, such as horses, cows, goats, sheep,dogs, chickens, rabbits, mice, and rats.

[0102] The immunogenicity of a zFGF5 polypeptide may be increasedthrough the use of an adjuvant, such as alum (aluminum hydroxide) orFreund's complete or incomplete adjuvant. Polypeptides useful forimmunization also include fusion polypeptides, such as fusions of zFGF5or a portion thereof with an immunoglobulin polypeptide or with maltosebinding protein. The polypeptide immunogen may be a full-length moleculeor a portion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

[0103] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting only non-human CDRs onto human framework andconstant regions, or by incorporating the entire non-human variabledomains (optionally “cloaking” them with a human-like surface byreplacement of exposed residues, wherein the result is a “veneered”antibody). In some instances, humanized antibodies may retain non-humanresidues within the human variable region framework domains to enhanceproper binding characteristics. Through humanizing antibodies,biological half-life may be increased, and the potential for adverseimmune reactions upon administration to humans is reduced. Alternativetechniques for generating or selecting antibodies useful herein includein vitro exposure of lymphocytes to zFGF5 protein or peptide, andselection of antibody display libraries in phage or similar vectors (forinstance, through use of immobilized or labeled zFGF5 protein orpeptide).

[0104] Antibodies are defined to be specifically binding if they bind toa zFGF5 polypeptide with a binding affinity (Ka) of 10⁶ M⁻¹ or greater,preferably 107 M⁻¹ or greater, more preferably 10⁸ M⁻¹ or greater, andmost preferably 10⁹ M⁻¹ or greater. The binding affinity of an antibodycan be readily determined by one of ordinary skill in the art (forexample, by Scatchard analysis).

[0105] A variety of assays known to those skilled in the art can beutilized to detect antibodies which specifically bind to zFGF5 proteinsor peptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant zFGF5protein or peptide.

[0106] Antibodies to zFGF5 may be used for tagging cells that expresszFGF5; to target another protein, small molecule or chemical to hearttissue; for isolating zFGF5 by affinity purification; for diagnosticassays for determining circulating levels of zFGF5 polypeptides; fordetecting or quantitating soluble zFGF5 as marker of underlyingpathology or disease; in analytical methods employing FACS; forscreening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockzFGF5 mediated proliferation in vitro and in vivo. Antagonists will beuseful for inhibiting bone formation where such formation results inpremature closure of the growth plate, for example in craniosyntosis.zFGF5 has been identified in endothelial and smooth muscle cells byimmunocytochemistry. In addition, there is evidence that zFGF5 plays arole in chemotaxis of macrophage. Therefore, antagonists to zFGF5 couldbe useful for inhibition of restenosis and artherosclerosis.

[0107] Suitable direct tags or labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like; indirect tags or labels mayfeature use of biotin-avidin or other complement/anti-complement pairsas intermediates. Antibodies herein may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.

[0108] Molecules of the present invention can be used to identify andisolate receptors involved in cardiac myocyte, cardiac fibroblast, orcardiac progenitor cell proliferation. For example, proteins andpeptides of the present invention can be immobilized on a column andmembrane preparations run over the column (Immobilized Affinity LigandTechniques, Hermanson et al., eds., Academic Press, San Diego, Calif.,1992, pp.195-202). Proteins and peptides can also be radiolabeled(Methods in Enzymol., vol. 182, “Guide to Protein Purification”, M.Deutscher, ed., Acad. Press, San Diego, 1990, 721-737) or photoaffinitylabeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedanet al., Biochem. Pharmacol. 33:1167-1180, 1984) and specificcell-surface proteins can be identified.

[0109] Antagonists will be useful for inhibiting the proliferativeactivities of zFGF5 molecules, in cell types such as cardiac cells,including myocytes, fibroblasts and endothelial cells, osteoblasts andchondrocytes. Genes encoding zFGF5 polypeptide binding domains can beobtained by screening random peptide libraries displayed on phage (phagedisplay) or on bacteria, such as E. coli. Nucleotide sequences encodingthe polypeptides can be obtained in a number of ways, such as throughrandom mutagenesis and random polynucleotide synthesis. These randompeptide display libraries can be used to screen for peptides whichinteract with a known target which can be a protein or polypeptide, suchas a ligand or receptor, a biological or synthetic macromolecule, ororganic or inorganic substances. Techniques for creating and screeningsuch random peptide display libraries are known in the art (Ladner etal., U.S. Pat. No:5,223,409; Ladner et al., U.S. Pat. No:4,946,778;Ladner et al., U.S. Pat. No:5,403,484 and Ladner et al., U.S. Pat.No:5,571,698) and random peptide display libraries and kits forscreening such libraries are available commercially, for instance fromClontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), NewEngland Biolabs, Inc. (Beverly, Mass.) and Pharmacia LKB BiotechnologyInc. (Piscataway, N.J.). Random peptide display libraries can bescreened using the zFGF5 sequences disclosed herein to identify proteinswhich bind to zFGF5. These “binding proteins” which interact with zFGF5polypeptides may be used for tagging cells; for isolating homologpolypeptides by affinity purification; they can be directly orindirectly conjugated to drugs, toxins, radionuclides and the like.These binding proteins can also be used in analytical methods such asfor screening expression libraries and neutralizing activity. Thebinding proteins can also be used for diagnostic assays for determiningcirculating levels of polypeptides; for detecting or quantitatingsoluble polypeptides as marker of underlying pathology or disease. Thesebinding proteins can also act as zFGF5 “antagonists” to block zFGF5binding and signal transduction in vitro and in vivo. These anti- zFGF5binding proteins would be useful for inhibiting expression of geneswhich result in proliferation or differentiation. Such anti-zFGF5binding proteins can be used for treatment, for example, inrhabdomyosarcoma, cardiac myxoma, bone cancers of osteoblast origin, anddwarfism, arthritis, ligament and cartilage repair, alone or combinationwith other therapies.

[0110] The molecules of the present invention will be useful forproliferation of cardiac tissue cells, such as cardiac myocytes,myoblasts or progenitors; skeletal myocytes or myoblasts and smoothmuscle cells; chondrocytes; endothelial cells; adipocytes andosteoblasts in vitro. For example, molecules of the present inventionare useful as components of defined cell culture media, and may be usedalone or in combination with other cytokines and hormones to replaceserum that is commonly used in cell culture. Molecules of the presentinvention are particularly useful in specifically promoting the growthand/or development of myocytes in culture, and may also prove useful inthe study of cardiac myocyte hyperplasia and regeneration.

[0111] The polypeptides, nucleic acid and/or antibodies of the presentinvention may be used in treatment of disorders associated with heartdisease, i.e., myocardial infarction, coronary artery disease,congestive heart failure, hypertrophic cardiomyopathy, myocarditis,congenital heart defects and dilated cardiomyopathy. Molecules of thepresent invention may also be useful for limiting infarct size followinga heart attack, promoting angiogenesis and wound healing followingangioplasty or endarterectomy, to develop coronary collateralcirculation, for revascularization in the eye, for complications relatedto poor circulation such as diabetic foot ulcers, for stroke, followingcoronary reperfusion using pharmacologic methods and other indicationswhere angiogenesis is of benefit. Molecules of the present invention maybe useful for improving cardiac function, either by inducing cardiacmyocyte neogenesis and/or hyperplasia, by inducing coronary collateralformation, or by inducing remodelling of necrotic myocardial area.

[0112] An ischemic event is the disruption of blood flow to an organ,resulting in necrosis or infarct of the non-perfused region.Ischemia-reperfusion is the interruption of blood flow to an organ, suchas the heart or brain, and subsequent restoration (often abrupt) ofblood flow. While restoration of blood flow is essential to preservefunctional tissue, the reperfusion itself is known to be deleterious. Infact, there is evidence that reperfusion of an ischemic area compromisesendothelium-dependent vessel relaxation resulting in vasospasms, and inthe heart compromised coronary vasodilation, that is not seen in anischemic event without reperfusion (Cuevas et al., Growth Factors15:29-40, 1997). Both ischemia and reperfusion are importantcontributors to tissue necrosis, such as a myocardial infarct or stroke.The molecules of the present invention will have therapeutic value toreduce damage to the tissues caused by ischemia or ischemia-reperfusionevents, particularly in the heart or brain.

[0113] Molecules of the present invention for be useful for thetreatment of injuries to the central nervous system. In particular,pharmaceutical compositions of zFGF5 will be useful in the treatment ofischemic events, such as stroke. The effects of zFGF5 compositions inischemic cerebrovascular disease (i.e., stroke) have been demonstratedusing a middle cerebral artery occlusion model of stroke in mice (HuangZ et al., Am J Physiol 272: H1401-H1405, 1996). As measured bydensitometry, administration of zFGF5 resulted in a 50% reduction ininfarct volume over vehicle in animals where cerebral blood flow hadbeen reduced by 75%, after transient occlusion of the anteriorcommunicating and middle cerebral arteries. Reduction in infarct volumewas greater using zFGF5 than vehicle, and was dose dependent. Theadministration of zFGF5 did not affect arterial blood pressure, _(p)O₂,pCO₂, or pH of the animals.

[0114] Generally, administration of drugs intended to limit damagecaused by ischemia and reperfusion are administered within hours ofdiagnosis of the ischemic event. The pharmaceutical compositioncomprising zFGF5 may be given by intra-arterially, intravenously, orintracerebrovascularly, or other methods for administration asdetermined by one skilled in the art. When determining efficacy of atherapeutic treatment for stroke, various tests are used clinically.Generally, these tests are directed toward evaluation of neurologicalfunction and patient progress toward recovery from neurologicaldeficiencies. The effects of stroke severity and recovery can bedetermined using scales designed to measure either impairment ordisability. Impairment is measured as the effect of the disease at theorgan level. Impairment scales for stroke can include measurement of armand leg motor function, speech, consciousness, facial paresis, muscletone, orientation, and reflexes. Disability scales measure limitationsin daily living or performance at a general level. Disability scales caninclude the patient's ability to walk, dress, feed themselves, bowel andbladder control and groom themselves. Outcomes can be measured atpredetermined increments, for example, 1, 3, 6, and 12 months. Twostroke scales that are used, particularly in clinical investigation, arethe Rankin stroke scale and Barthel scale. The Rankin scale uses fivegrades: 1=“no significant disability”; 2=“slight disability”;3=“moderate disability”; 4=“moderately severe disability”; and 5=“severedisability”. The Barthel scale consists of ten activities that include:feeding, transfer, grooming, toilet use, bathing, mobility, stairclimbing, dressing and continence for bowel and bladder. The score rangeis 0-100, with 100 being maximal or normal function. Other measurementsfor recovery from stroke include the NIH stoke scale, Mathew scale andScandinavian scale. See, e.g., Ginsberg and Bogousslavsky, eds.Cardiovascular Disease: Pathophysiology, Diagnosis and Management.Maiden, MA. Blackwell Science, 1998; and Duncan et al., Stroke30:2131-2140, 1999. zFGF5 induced coronary collateral development ismeasured in rabbits, dogs or pigs using models of chronic coronaryocclusion (Landau et al., Amer. Heart J. 29:924-931, 1995; Sellke etal., Surgery 120(2):182-188, 1996 and Lazarous et al., 1996, ibid.)zFGF5 benefits for treating stroke is tested in vivo in rats utilizingocclusion of the middle cerebral artery or carotid artery occlusion andmeasuring histological changes, as well as maze performance (Gage etal., Neurobiol. Aging 9:645-655, 1988). zFGF5 efficacy in hypertensionis tested in vivo utilizing spontaneously hypertensive rats (SHR) forsystemic hypertension (Marche et al., Clin. Exp. Pharmacol. Physiol.Suppl. 1:S114-116, 1995). Other therapeutic uses for the presentinvention include induction of skeletal muscle neogenesis and/orhyperplasia, kidney regeneration and/or for treatment of systemic andpulmonary hypertension.

[0115] Additional uses for zFGF5 compositions include treatment ofneuronal degenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington'sdisease; and traumatic injuries to brain or spinal cord, nervous systemtumors, infection, dementia, epilepsy, and peripheral nerve injury.

[0116] Molecules of the present invention can be used to target thedelivery of agents or drugs to the heart. For example, the molecules ofthe present invention will be useful limiting expression to the heart,by virtue of the tissue specific expression directed by the zFGF5promoter. For example, heart-specific expression can be achieved using azFGF5-adenoviral discistronic construct (Rothmann et al., Gene Therapy3:919-926, 1996). In addition, the zFGF5 polypeptides can be used torestrict other agents or drugs to heart tissue by linking zFGF5polypeptides to another protein (Franz et al., Circ. Res. 73:629-638,1993) by linking a first molecule that is comprised of a zFGF5 homologpolypeptide with a second agent or drug to form a chimera. Proteins, forinstance antibodies, can be used to form chimeras with zFGF5 moleculesof the present invention (Narula et al., J. Nucl. Cardiol. 2:26-34,1995). Examples of agents or drugs include, but are not limited to,bioactive-polypeptides, genes, toxins, radionuclides, small moleculepharmaceuticals and the like. Linking may be direct or indirect (e.g.,liposomes), and may occur by recombinant means, chemical linkage, strongnon-covalent interaction and the like.

[0117] Polynucleotides encoding zFGF5 polypeptides are useful withingene therapy applications where it is desired to increase or inhibitzFGF5 activity. If a mammal has a mutated or absent zFGF5 gene, thezFGF5 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a zFGF5 polypeptide is introduced in vivo ina viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al.,J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

[0118] In another embodiment, a zFGF5 gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication No. WO 95/07358, published Mar. 16,1995 by Dougherty et al.; and Kuo et al., Blood 82:845, 1993.Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl.Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types in a tissuewith cellular heterogeneity, such as the heart, brain, lungs or liver.Lipids may be chemically coupled to other molecules for the purpose oftargeting. Targeted peptides (e.g., hormones or neurotransmitters),proteins such as antibodies, or non-peptide molecules can be coupled toliposomes chemically.

[0119] It is possible to remove the target cells from the body; tointroduce the vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

[0120] Antisense methodology can be used to inhibit zFGF5 genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a zFGF5-encodingpolynucleotide (e.g., a polynucleotide as set froth in SEQ ID NO: 1) aredesigned to bind to zFGF5-encoding mRNA and to inhibit translation ofsuch mRNA. Such antisense polynucleotides are used to inhibit expressionof zFGF5 polypeptide-encoding genes in cell culture or in a subject.

[0121] The present invention also provides reagents which will find usein diagnostic applications. For example, the zFGF5 gene, a probecomprising zFGF5 DNA or RNA or a subsequence thereof can be used todetermine if the zFGF5 gene is present on chromosome 5 and if a mutationin the zFGF5 gene locus has occurred including, but not limited to,aneuploidy, gene copy number changes, insertions, deletions, restrictionsite changes and rearrangements. Such aberrations can be detected usingpolynucleotides of the present invention by employing molecular genetictechniques, such as restriction fragment length polymorphism (RFLP)analysis, short tandem repeat (STR) analysis employing PCR techniques,and other genetic linkage analysis techniques known in the art (Sambrooket al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).

[0122] Mice engineered to express the zFGF5 gene, referred to as“transgenic mice,” and mice that exhibit a complete absence of zFGF5gene function, referred to as “knockout mice,” may also be generated(Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature366:740-42, 1993; Capecchi, M.R., Science 244: 1288-1292, 1989;Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 1986). For example,transgenic mice that over-express zFGF5, either ubiquitously or under atissue-specific or tissue-restricted -promoter can be used to askwhether over-expression causes a phenotype. For example, over-expressionof a wild-type zFGF5 polypeptide, polypeptide fragment or a mutantthereof may alter normal cellular processes, resulting in a phenotypethat identifies a tissue in which zFGF5 expression is functionallyrelevant and may indicate a therapeutic target for the zFGF5, itsagonists or antagonists. For example, a preferred transgenic mouse toengineer is one that over-expresses the zFGF5 (approximately amino acidresidue 28 to residue 207 of SEQ ID NO:2). Moreover, suchover-expression may result in a phenotype that shows similarity withhuman diseases. Similarly, knockout zFGF5 mice can be used to determinewhere zFGF5 is absolutely required in vivo. The phenotype of knockoutmice is predictive of the in vivo effects of a zFGF5 antagonist, such asthose described herein, may have. These mice may be employed to studythe zFGF5 gene and the protein encoded thereby in an in vivo system, andcan be used as in vivo models for corresponding human diseases.

[0123] In one embodiment of the present invention, a compositioncomprising zFGF5 protein is used as a therapeutic agent to enhanceosteoblast-mediated bone formation. The compositions and methods usingthe compositions of the invention may be applied to promote the repairof bone defects and deficiencies, such as those occurring in closed,open and non-union fractures; to promote bone healing in plasticsurgery; to stimulate bone ingrowth into non-cemented prosthetic jointsand dental implants; in the treatment of periodontal disease anddefects; to increase bone formation during distraction osteogenesis; andin treatment of other skeletal disorders that may be treated bystimulation of osteoblastic activity, such as osteoporosis andarthritis. De novo bone formation provided by the methods of the presentinvention will have use in repair of congenital, trauma-induced,oncologic resection of bone or healing bone following radiation-inducedosteonecrosis (Hart et al, Cancer 37:2580-2585, 1976). The methods ofthe present invention may also find use in plastic surgery.

[0124] The molecules of the present invention provide a method forstimulating the proliferation of chondrocytes, in particulardifferentiated chondrocytes capable of inducing specialized cellfunctions, normally associated with terminally differentiated cells.When zFGF5 was administered locally to chondrocytes isolated fromarticular cartilage, cartilage explants, proliferation of the cells andconcomitant synthesis of glycosaminoglycans was increased. Additionally,an increase in cartilaginous tissue in ear has been measured in miceears injected with adenovirus expressing zFGF5. These results indicatethat zFGF5 can play a therapeutic role in maintaining or repairingcartilaginous tissue, such as joints damaged by osteoarthritis,rheumatoid arthritis or traumatic injury.

[0125] ZFGF5 have been shown to increase cartilage deposition both invivo and in vitro. Generation of hyaline cartilage, elastic cartilage,and fibrocartilage are valuable both as a therapeutic and as componentfor biological matrices. zFGF5 compositions will be useful in treatingarticular cartilage defects in synovial joints that are due toage-related superficial fibrillation, cartilage degeneration due toosteoarthritis, and focal chondral and osteochondral defects due toinjury or disease. ZFGF5 compositions will also be useful for treatingjoint disease caused by osteochondritis dissecans and degenerative jointdisease. In the field of reconstructive and plastic surgery, zFGF5compositions will be useful for autogenous or allogenic cartilageexpansion and transfer for reconstruction of extensive tissue defects.Expansions of cells and induction of elastic cartilage production willbe useful for generation and repair of ear and nose tissue.

[0126] ZFGF5 compositions can be applied by direct injection into thesynovial fluid of the joint or directly into the defect, either alone orcomplexed with a suitable carrier for extended release of protein. ZFGF5can also be used to expand chondrocyte populations in culture forautogenous or allogenic chondrocyte transplantation. In theseprocedures, for example, chondrocytes can be harvested arthroscopicallyfrom an uninjured minor load-bearing area of the damaged joint, and canbe cultured in the presence of zFGF5 compositions to increase the numberof cells prior to transplantation. The expanded cultures will then beadmixed with zFGF5 compositions and placed in the joint space ordirectly into the defect. ZFGF5 compositions can be used in combinationwith periosteal or perichondrial grafts that contain cells that can formcartilage and/or help to hold the transplanted chondrocytes or theirprecursor cells in place. ZFGF5 compositions can be used to repaircartilage damage in conjunction with lavage of the joint, stimulation ofbone marrow, abrasion arthroplasty, subchondral drilling, ormicrofracture of the subchondral bone.

[0127] For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,administration according to conventional methods. Intravenousadministration will be by bolus injection or infusion over a typicalperiod of one to several hours. In general, pharmaceutical formulationswill include a zFGF5 protein in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, extend half-life, etc. Methods offormulation are well known in the art and are disclosed, for example, inRemington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co.,Easton Pa., 1990, which is incorporated herein by reference. Therapeuticdoses will generally be in the range of 0.1 to 100 μg/kg of patientweight per day, preferably 0.5-20 μg/kg per day, with the exact dosedetermined by the clinician according to accepted standards, taking intoaccount the nature and severity of the condition to be treated, patienttraits, etc. Determination of dose is within the level of ordinary skillin the art. The proteins may be administered for acute treatment, overone week or less, often over a period of one to three days or may beused in chronic treatment, over several months or years.

[0128] In other embodiments, a pharmaceutical zFGF5 composition willcomprise a formulation for timed-release of the protein. Time-releaseformulations generally include a monolithic delivery device comprisingbiocompatible solutions, gels, pastes, and putties in a matrix, in whichthe composition is entrapped or dissolved. Release from such atimed-release composition occurs by diffusion through the matrix and/orerosion of the matrix. A reservoir system, where the pharmaceuticalcomposition diffuses through a membrane, may also be used.

[0129] Delivery devices can include, but are not limited to, medicaldevices; sutures; and solid matrices, such as collagen sponges;ethylene-vinyl acetate copolymers; and acrylonitrile-vinyl chloridecopolymers. Liquid delivery devices would include, for example,hydrogels. Examples of biodegradable polymers include: poly(DL-lactide),poly(DL-lactice co-glycolide), poly(DL-lactide-co-caprolactone) andpolyanhydrides and ATRIGEL® (Atrix Laboratories, Fort Collins, Colo.).Another delivery device can include poloxamers, for example, Pluronic®F-127 (BASF, Parsippany, N.Y.), which are liquid at room temperature andform gels at 37° C. Additional delivery devices include negativelycharged macromolecules such as hyaluronic acid, sulfated proteoglycans,B-cyclodextrin tetradecasulphate; or hydroxyapatite, alginatemicrospheres, and methylcellulose. For review of delivery systems forgrowth factors, see, e.g., Minmi, Biomaterials 18:1201-1225, 1997; andLanger et al., J. Cell. Biochem. 45:340-345, 1991.

[0130] In one embodiment, a therapeutically effective amount of zFGF5 isan amount sufficient to produce a clinically significant change inmyocyte proliferation, heart function, brain function, bone formation orincreases in specific cell types associated with mesenchymal stem cellsand progenitors for myocytes, osteoblasts and chondrocytes. Inparticular, a clinically significant improvement in cardiac performancemay be an increase in the number of myocytes or myocyte progenitorcells. Improvements in cardiac performance can be determined by methodswell known and accepted by clinicians and those skilled in the art. Suchdeterminations include, but are not limited to, measuring the leftventricular ejection fraction, prior to, and after administration ofzFGF5 molecules, and determining at least a 5% increase, preferably 10%or more, in the total ejection fraction, increases in −dP/dt or +dP/dt,greater exercise tolerance, a decrease in vascular resistance, andincreased blood flow to the heart. A reduction in symptoms may also beindication of a significant improvement in cardiac performance, andinclude, for example, reduction in angina pectoris, breathlessness, legswelling, heart or respiratory rates, edema, fatigue and weakness.

[0131] Thus, in summary, certain aspects the present invention includes:Methods of reducing infarct volume in a mammal diagnosed as having acerebrovascular ischemic stroke comprising: (1) determining infarctvolume in the mammal; (2) administering a pharmaceutical compositioncomprising a polypeptide that is at least 80% identical to an amino acidsequence as shown in SEQ ID NO: 2 from amino acid residue 28 (Glu) toamino acid residue 175 (Met) sufficient to reduce infarct volume; (3)determining infarct volume in the mammal; and (4) comparing the infarctvolume of step 1 to step 4. In further embodiments the pharmaceuticalcomposition is a polypeptide that is at least 80% identical to the aminoacid sequence as shown in SEQ ID NO: 2 from residue 28 (Glu) to 196(Lys) and is a polypeptide is the amino acid sequence as shown in SEQ IDNO: 2 from residue 28 (Glu) to residue 196 (Lys).

[0132] In another aspect, the present invention includes methods fortreating a patient who has suffered an injury to the central nervoussystem, comprising administering to the patient a pharmaceuticalcomposition comprising a fibroblast growth factor (FGF) homologpolypeptide, wherein said polypeptide comprises a sequence of aminoacids that is at least 80% identical to the amino acid sequence as shownin SEQ ID NO: 2 from amino acid residue 28 (Glu) to 175 (Met), in amountsufficient to improve functional recovery in the patient. In otherembodiments, the injury to the central nervous system is an ischemicevent such as a stroke. In further embodiments, improved functionalrecovery is defined as a patient score of at least 3 on a Rankin strokescale, or is defined as a patient score of lower than 75 on a Barthel'sscale. Furthermore, certain embodiment include methods wherein saidpolypeptide encoded by said polynucleotide is at least 90% identical tothe amino acid sequence as shown in SEQ ID NO: 2 from amino acid residue28 (Glu) to residue 196 (Lys) or is at least 80% identical to the aminoacid sequence as shown in SEQ ID NO: 2 from amino acid residue 28 (Glu)to residue 207 (Ala).

[0133] In another aspect, the present invention can be summarized asmethods of increasing cartilage deposition in a patient comprising:administering a pharmaceutical composition comprising a fibroblastgrowth factor (FGF) homolog polypeptide to a fibrocartilage, hyaline, orelastic cartilage injury in said patient, wherein said polypeptidecomprises a sequence of amino acids that is at least 80% identical tothe amino acid sequence as shown in SEQ ID NO: 2 from amino acid residue28 (Glu) to 175 (Met). Furthermore, additional embodiments include whenthe administration of the composition is selected from the groupconsisting of intracartilaginous administration; intraarticular,intravenous and intramuscular administration; and topicaladministration.

[0134] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1

[0135] Extension of EST Sequence

[0136] Scanning of a translated DNA database using a query for growthfactors resulted in identification of an expressed sequence tag (EST)sequence found to be a novel member of the FGF family, and designatedzFGF5.

[0137] Oligonucleotide primers ZC11676 (SEQ ID NO: 3) and ZC11677 (SEQID NO: 4) were designed from the sequence of an expressed sequence tag(EST). The primers were used for priming internally within the EST, andwhen PCR was performed using MARATHON READY cDNA (Clontech, Palo Alto,Calif.) from adult heart tissue as template in polymerase chain reaction(PCR).

[0138] The conditions used for PCR were 1 cycle at 94° C. for 90seconds, 35 cycles at 94° C. for 15 seconds; 68° C. for 1 minute;followed by 1 cycle for 10 minutes at 72° C. and 4° C. incubationperiod. The PCR reaction recreated 160 bp of the EST sequence, andconfirmed that EST sequence was correct.

[0139] Other libraries that could be amplified with the oligonucleotideprimers included skeletal muscle, lung, stomach, small intestine andthyroid.

Example 2

[0140] Tissue Distribution

[0141] Northerns were performed using Human Multiple Tissue Blots fromClontech (Palo Alto, Calif.). The 160 bp DNA fragment described inExample 1 was electrophoresed on a 1% agarose gel, the fragment waselectroeluted, and then radioactively labeled using a random primingMEGAPRIME DNA labeling system (Amersham, Arlington Heights, Ill.)according to the manufacturer's specifications. The probe was purifiedusing a NUCTRAP push column (Stratagene Cloning Systems, La Jolla,Calif.). EXPRESSHYB (Clontech, Palo Alto, Calif.) solution was used forprehybridization and as a hybridrizing solution for the Northern blots.Hybridization took place overnight at 8° C., and the blots were thenwashed in 2X SSC and 0.05% SDS at RT, followed by a wash in 0.1X SSC and0.1% SDS at 50° C. A single band was observed at approximately 2.0 kb.Signal intensity was highest for adult heart with relatively lessintense signals in skeletal muscle and stomach. Dot blots were probedessentially as described above, confirming that expression for humanzFGF5 was highest in heart tissue followed by lung and skeletal muscle.

Example 3

[0142] Assay for In Vitro Activity of zFGF5

[0143] A.

[0144] The mitogenic activity of zFGF5 is assayed using cell lines andcells from a primary culture. Conditioned medium from cells expressingthe recombinant protein and/or purified protein is added to cultures ofthe following cell lines: NIH 3T3 fibroblast (ATCC No. CRL-1658), CHH-1chum heart cells (ATCC No. CRL-1680), H9c2 rat heart myoblasts (ATCC No.CRL-1446), Shionogi mammary carcinoma cells (Tanaka et al., 1992, ibid.)and LNCaP.FGC adenocarcinoma cells. Freshly isolated cells useful fortesting the proliferative activity of zFGF5 include: cardiacfibroblasts, cardiac myocytes, skeletal myocytes and human umbilicalvein endothelial cells.

[0145] Mitogenic activity is assayed by measurement of ³H-thymidineincorporation based on the method of Raines and Ross (Meth. Enzymology109:749-773, 1985). Briefly, quiescent cells are plated cells at adensity of 3 x 10⁴ cells/ml in an appropriate medium. A typical growthmedium is Dulbecco's Growth Medium (GIBCO-BRL, Gaithersburg, Md.)containing 10% fetal calf serum (FCS). The cells are cultured in 96-wellplates and allowed to grow for 3-4 days. The growth medium is removed,and 180 μl of DFC (Table 5) containing 0.1% FCS is added per well. Halfthe wells have zFGF5 protein added to them and the other half are anegative control, without zFGF5. The cells are incubated for up to 3days at 37° C. in 5% CO₂, and the medium is removed. One hundredrmicroliters of DFC containing 0.1% FCS and 2 μCi/ml ³H-thyrmidine isadded to each well, and the plates are incubated an additional 1-24hours at 37° C . The medium is aspirated off, and 150 μl of trypsin isadded to each well. The plates are incubated at 37° C. until the cellsdetached (at least 10 minutes). The detached cells are harvested ontofilters using an LKB Wallac 1295-001 Cell Harvester (LKB Wallac,Pharmacia, Gaithersburg, Md.). The filters are dried by heating in amicrowave oven for 10 minutes and counted in an LKB Betaplate 1250scintillation counter (LKB Wallac) as described by the supplier. TABLE 5250 ml Dulbecco's Modified Eagle's Medium (DMEM, Gibco-BRL) 250 ml Ham'sF12 medium (Gibco-BRL) 0.29 mg/ml L-glutamine (Sigma, St. Louis, MO) 1mM sodium pyruvate (Sigma, St. Louis, MO) 25 mM Hepes (Sigma, St. Louis,MO) 10 μg/ml fetuin (Aldrich, Milwaukee, WI) 50 μg/ml insulin(Gibco-BRL) 3 ng/ml selenium (Aldrich, Milwaukee, WI) 20 μg/mltransferrin (JRH, Lenexa, KS)

[0146] B.

[0147] Hearts were isolated from 1 day old neonatal mice and thendisrupted by repeat collagenase digestions, following the protocol ofBrand et al., (J. Biol. Chem. 268:11500-11503, 1993). Individualmyocytes were isolated over a Percoll gradient, and 2 ml were plated in6 well tissue culture dishes at 0.5×10⁶ cells/ml. Three days later thewells were washed 3 times with PBS without calcium or magnesium, andrefed with 1 ml serum free medium (Table 6). The wells were inoculatedwith 10ll particles AdCMV-zFGF5 per well or AdCMV-GFP (green fluorescentprotein) as a control, and incubated at 37° C. for 8 hours. The wellswere then washed again 3 times with PBS without calcium or magnesium,and then refed with 2 mls serum free media.

[0148] Within 48 hours after inoculation with the AdCMV-zFGF5, thecultured myocytes have ceased to beat and have undergone a morphologicalteration, while the wells inoculated with the AdCMV-GFP continued tobeat spontaneously and are unaffected morphologically by theinoculation. Wells inoculated with AdCMV-zFGF5 also contained, after 48,hours, a confluent layer of viable, non-adherent cells, without any lossin confluence of the adherent myocyte layers, indicating theproliferative activity of the adCMV-zFGF5 on cultured murine myocytes.TABLE 6 DMEM Ham's Nutrient Mixture F12 (Gibco-BRL; 1:1 mixture withDMEM) 17 mM NaHCO₃ (Sigma) 2 mM L-glutamine (Sigma) 1% PSN (Sigma) 1μg/ml insulin 5 μg/ml transferrin 1 nM LiCl (Sigma) 1 nM selenium 25μg/ml ascorbic acid (Sigma) 1 nM thyroxine (Sigma)

[0149] C.

[0150] zFGF5 fused to a maltose binding protein (MBP), as described inExample 9A and purified as described in Example 10, was added tomyocytes (Example 3B) at a concentration of 0.1 ng/ml. MBP-zFGF5 wasshown to stimulate proliferation of myocytes, as well.

Example 4

[0151] Assay for Ex Vivo Activity of zFGF5

[0152] Cardiac mitogenesis is measured ex vivo by removing entire heartsfrom neonatal or 8-week old mice or rats. The excised heart is placed inJoklik's (Sigma, St. Louis, Mo.) or Dulbecco's medium. at 37° C., 5% CO₂for 4-24 hours. During the incubation period zFGF5 polypeptide is addedat a concentration range of 1 pg/ml to 100 μg/ml. Negative controls areusing buffer only. ³H-thymidine is added and the samples are incubatedfor 1-4 hours, after which the heart is sectioned and mitogenesis isdetermined by autoradiography. Sections are used for histomorphometry todetermine the nuclei/cytoplasmic volume (McLaughlin, Am. J. Physiol.271:R122-R129, 1996.)

[0153] Alternatively, the heart was lyophilized and resuspended in 1 ml0.1 N NaOH. The DNA was precipitated using ice cold 10% trichloroaceticacid (TCA). The supernatant was added to 9 ml scintillation fluid tomeasure non-specific ³H-thymidine incorporation. The resulting pelletwas resuspended in 1 ml BTS-450 tissue solubilizer (Beckman, Fullerton,Calif.) and added to 9 ml of scintillation fluid to measure specific,DNA incorporation of ³H-thymidine.

[0154] Left and right ventricles were isolated from 1 day old CD-1 mice(Jackson Labs, Bar Harbor, Me.), and incubated for 4 hours with 3 ng/mlzFGF5Hep2 (n=13; see Example 10) or control (n=10). ³H-thymidine wasadded for 1 hour. The ventricles were washed several times and thenhomogenized in 1 ml Joklik's medium. The resulting homogenate was addedto 9 ml scintillation cocktail and analyzed for total ³H-thymidineuptake and DNA incorporation.

[0155] zFGF5-Hep2 increased ³H-thymidine uptake and incorporation in DNA2.068+0.489 fold over control, indicating that zFGF5 is mitogenic for acardiac cell.

Example 5

[0156] Assay for In Vivo Activity of zFGF5

[0157] The proliferative effects of zFGF5 are assayed in vivo usingtwo-week old neonatal rats and/or two-month old adult rats. The rats areinjected intraperiocardially either acutely or chronically.

[0158] A.

[0159] Neonatal rats are treated with zFGF5 for 1 to 14 days over a doserange 35 of 50 ng/day to 100 μg/day. After treatment, the effects ofzFGF5 versus the sham-treated animals is evaluated by measuringincreased cardiac weight, improved in vivo and ex vivo left ventricularfunction, and by increased cardiac nuclear to cytosolic volumefractions, that are determined histomorphometrically.

[0160] B.

[0161] Rats with cardiomyopathy induced by chronic catecholamineinfusion, by coronary ligation or for models of cardiomyopathy such asthe Syrian Cardiomyopathic hamster (Sole et al., Amer. J. Cardiol.62(11):20G-24G, 1988) are also used to evaluate the effects of zFGF5 oncardiac function and tissue.

[0162] To induce cardiomyopathy using catecholamine, 7-8 week old ratsare infused continuously with epinephrine for 2 weeks via osmoticminipumps implanted subcutaneously between their shoulder blades. Theepinephrine infusion results in an increase in the left ventricularfibrotic lesion score from 0.005+0.005 to 2.11+0.18 scale from 0-3);increased left ventricular myocyte cell width from 17.36+0.46 μm to23.05±0.62 μm; and negligible left ventricular papillary musclecontractile responses to isoproterenol (0.2 vs 1.1 grams tensioncompared to saline-infused rats. After the two week treatment period,the rats are injected intraperiocardially daily with either vehicle,zFGF5, bFGF, IGF-I or IGF-II for up to 14 days. The rats are sacrificedand histomorphometry and histocytochemistry are performed.

[0163] Rats, treated as described above, are also evaluated at the endof the cathecholamine treatment, and again after growth factortreatment, where cardiac regeneration is measured as decreased leftventricular fibrotic lesion scores, reduced myocyte cell width andincreased left ventricular papillary contractile responses toisoproterenol.

Example 6

[0164] Chromosomal Mapping of zFGF5

[0165] ZFGF5 was mapped to chromosome 5 using the commercially availableversion of the Whitehead Institute/MIT Center for Genome Research's“GeneBridge 4 Radiation Hybrid Panel” (Research Genetics, Inc.,Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panel contains DNAssuitable for PCR use from each of 93 radiation hybrid clones, plus twocontrol DNAs (the HFL donor and the A23 recipient). A publicly availableWWW server allows mapping relative to the Whitehead Institute/MIT Centerfor. Genome Research's radiation hybrid map of the human genome (the“WICGR” radiation hybrid map) which was constructed with the GeneBridge4 Radiation Hybrid Panel.

[0166] For the mapping of zFGF5 with the “GeneBridge 4 RH Panel”, 25 μlreactions were set up in a 96-well microtiter plate (Stratagene, LaJolla, Calif.) and used for PCR in a “RoboCycler Gradient 96” thermalcycler (Stratagene). Each of the 95 PCR reactions consisted of 2.5 μl5OX “Advantage KlenTaq Polymerase Mix” (Clontech), 2 μl dNTPs mix (2.5mM each; Perkin-Elmer, Foster City, Calif.), 1.25 μl sense primer,ZC11677 (SEQ ID NO: 4) 1.25 μl antisense primer, ZC12053 (SEQ ID NO: 5).

[0167] 2.5 μL “RediLoad” (Research Genetics, Inc), 0.5 μl “AdvantageKlenTaq Polymerase Mix” (Clontech Laboratories, Inc.), 25 ng of DNA froman individual hybrid clone or control and ddH20 for a total volume of 25μl. The reactions were overlaid with an equal amount of mineral oil andsealed. The PCR cycler conditions were as follows: an initial 1 cycle of4 minutes at 94° C., 35 cycles of 1 minute at 94° C., 1.5 minuteannealing at 66° C. and 1.5 minute extension at 72° C., followed by afinal 1 cycle extension of 7 minutes at 72° C. The reactions wereseparated by electrophoresis on a 3% NuSieve GTG agarose gel (FMCBioproducts, Rockland, ME).

[0168] The results showed that zFGF5 maps 541.12 cR from the top of thehuman chromosome 5 linkage group on the WICGR radiation hybrid map.Relative to the centromere, its nearest proximal marker was WI-16922 andits nearest distal marker was WI-14692. The use of surrounding CHLC mapmarkers also helped position zFGF5 in the 5q34-q35 region on the CHLCchromosome 5 version v8c7 integrated marker map (The Cooperative HumanLinkage Center).

Example 7

[0169] zFGF5 Effects on Bone

[0170] A. Adenovirus Expressed zFGF5

[0171] An adenovirus vector containing the CDNA for zFGF5 wasconstructed using methods described by Becker et al. (Methods in CellBiology 43:161-189, 1994). Briefly, the CDNA for zFGF5 (as shown in SEQID NO: 1) was cloned as a Xba I-Sal I fragment into pACCMV (Gluzman etal., In Eucaryotic Viral Vectors, Gluzman (eds.) pp.187-192, Cold SpringHarbor Press, Cold Springs Harbor N.Y., 1982). The pACCMV vectorcontains part of the adenovirus 5 genome, the CMV promoter and an SV40terminator sequence. The plasmid containing the vector and cDNA insertwas cotransfected with a plasmid containing the the adenovirus 5 genome,designated pJM17, (McGrory et al., Virology 163:614-617, 1988) into 293cells (ATCC No. CRL-1573; American Type Culture Collection, Rockville,Md.), leading to a recombination event and the production of arecombinant adenovirus containing zFGF5, designated AdCMV-zFGF5. Thepresence of the zFGF5 cDNA was confirmed by PCR.

[0172] The adenovirus vector AdCMV-zFGF5 was used for gene tranfer invivo by intravenous injection of between 1×10¹¹ and 5×10¹¹particles/mouse. It has been shown that after intravenous injection, themajority of the virus targets the liver and very efficiently transduceshepatocytes (Herz et al., Proc. Natl. Acad. Sci. USA 90:2812-2816,1993). It has been demonstrated that the cells produce the proteinencoded by the cDNA, and in the case of secreted proteins, secret theminto the circulation. High levels of expression and physiologicaleffects have been demonstrated (Ohwada et al., Blood 88:768-774, 1996;Stevenson et al., Arteriosclerosis, Thrombosis and Vascular Biology,15:479-484, 1995; Setoguchi et al., Blood 84:2946-2953, 1994; andSakamoto et al., Proc. Nati. Acad. Sci. USA 91:12368-12372, 1994).

[0173] Six week old CD-1 mice (Jackson Labs, Bar Harbor, Me.) weretreated with adenovirus containing no cDNA insert (AdCMV-null) orAdCMV-zFGF5 either IV through the tail vein or intrapericardially (IPC).A total of 5×10¹¹ viral particles/100 μl/mouse were given. 14 days afterinjection, the animals were sacrificed, and tibias and femurs wereremoved without being separated to examine any potential inflammatoryresponse. The bones were fixed in 10% neutral buffered formalin andprocessed. They were decalcified in 5% formic acid with 10% sodiumcitrate, washed in water, dehydrated in a series of 70%-100% ethanol,cleared in xylene and embedded in paraffin. The specimens were cutlongitudinally through both tibial and femoral metaphyses and stainedwith hematoxylin and eosin for identification of bone cells. Osteoblastswere identified by central negative Golgi area and eccentric nucleus,while osteoclasts were identified by multinucleation, non-uniform shapeand the Howship's lacunae associated with these resorbing cells.

[0174] For bone histomorphometry, femur samples were chosen. Cancellousbone volume was not measured due to variation in the sampling site(i.e., femur samples were not sectioned exactly at the same plane).Three bone parameters were evaluated for histomorphometric changes.

[0175] 1. Number of endosteal osteoblasts: measured along the endostealsurface of cancellous bone at 180 X magnification in an area 1.22 mmproximal to the growth plate.

[0176] 2. Number of endosteal osteoclasts: measured along the endostealsurface of cancellous bone at 180 X magnification in an area 1.22 mmproximal to the growth plate.

[0177] 3. Growth plate width: measured every 72 μm at 90 X magnificationacross the entire growth plate except at the peripheral ends todetermine the growth plate activity.

[0178] Analyses of the data (mean ± SD, n=4-7/group) demonstrated thefollowing:

[0179] 1. There appeared to be no detectable inflammatory response atthe joint between tibia and femur. 2. AdCMV-zFGF5 given IV or IPC inmice significantly increased osteogenic activity in the distal femuralmetaphysis, when examined at 2 weeks. This stimulation of osteogenicactivity was indicated by:

[0180] a) significant increases in the number of endosteal osteoblastsin the cancellous bone of distal femurs following IV infusion or IPCinjection of AdCMV-zFGF5, 530% and 263%, respectively, when comparedwith their relative vector only controls; and

[0181] b) the observation of increased osteogenic tissues on the bonesurface, suggesting increased differentiation of bone marrow stromalcells toward the osteoblast lineage.

[0182] 3. The number of endosteal osteoclasts was not significantlyaffected by IV or IPC administration of AdCMV-zFGF5, when compared withtheir relative vector only controls.

[0183] 4. The growth plate width was significantly decreased by IVinfusion, but not IPC injection, of AdCMV-zFGF5, suggesting depressedgrowth plate activity following IV infusion. The differential effects ofAdCMV-zFGF5 administrations have not been elucidated.

[0184] These results suggest that zFGF5 is a strong mitogen forstimulation of osteoblast proliferation and that zFGF5 has the capacityto induce new bone formation.

[0185] Using essentially the same procedures described above in 7.A. QCTwas done on female CD-1 (Jackson Labs) that were injected with 1×10¹¹particles AdCMV-zFGF5 per mouse. The mice were sacrificed 30 days afterinjection and heart/tibial length ratios were increased compared tocontrols (injected with empty adenorvirus or saline). There were nodifferences between the groups in tibial lengths to account for thechange, nor were there differences in any other organ weights among thegroups. Thus, the indication is that zFGF5 adenovirus selectivelyincreases total bone density, trabecular bone density, and corticalthickness in the femur, as measured by QCT.

[0186] B. Effect of zFGF5 Pure Protein on Osteogenesis

[0187] An experimental pharmacology study was undertaken to evaluate theacute safety of zFGF5 over a large dose range. Individual maleSprague-Dawley rats were given a single bolus IV injection of zFGF5 viaan indwelling venous catheter at dose levels of 8 mg/kg, 40 mg/kg and 76mg/kg. Two additional animals were given vehicle alone. On day 14 postdose, these animals as well as two untreated male animals of the sameage were euthanized by C0₂, necropsied and tissues collected andpreserved in 10% buffered formalin. When present, the following tissueswere trimmed, processed, and examined microscopically from each animal:brain, liver, kidney, lung, heart, thymus, spleen, mesenteric lymphnode, pancreas, salivary gland, stomach, small intestine, largeintestine, testis, epididymis, urinary bladder, seminal vesicle,trachea, esophagus, thyroid, adrenal, pituitary, skeletal muscle, femur,and bone marrow.

[0188] Microscopically, the only test-related findings were in sectionsof the femur. The femurs from animals given the highest doses of zFGF5had evidence of hyperostosis and osteoblast hyperplasia. These findingswere accompanied by an apparent premature closing of the growth plate,seen as a loss of epiphysial cartilage, as well as an increaseddeposition of new bone along the diaphysis or shaft. There were no othertest-material related findings in any of the remaining tissues examined,nor were any significant changes noted in the CBCs or serum chemistries.There were no changes noted in the kidney similar to those reported forFGF-2. These results suggest that zFGF5 will be useful as an osteogenicfactor for treatment of fracture repair and osteoporosis and will beuseful as an osteogenic factor in reconstructive surgery.

Example 8

[0189] Effects of zFGF5 on Heart

[0190] As described in 7.B. CD-1 mice were given a single IV injectionof AdCMV-zFGF5, sacrificed after four weeks, and the heart/tibial lengthratios were found to be increased compared to empty adenovirus or salinetreated mice. The results showed that there were no differences betweenthe groups in tibial lengths to account for this change, nor were theredifferences in any other organ weights among the groups. This resultsuggests that AdCMV-zFGF5 selectively increased cardiac growth, whenadministered as an IV adenoviral construct.

Example 9

[0191] Expression of zFGF5

[0192] A. Construction of zFGF5-Encoding Plasmids

[0193] zFGF5, a fibroblast growth factor homolog, was expressed in E.coli using the MBP (maltose binding protein) fusion system from NewEngland Biolabs (NEB; Beverly, Mass.). In this system, the zFGF5 cDNAwas attached to the 3′ end of the malE gene to form an MBP-zFGF5 fusionprotein. Fusion protein expression was driven by the tac promoter;expression is “off” until the promoter is induced by addition of 1 mmolIPTG (isopropyl b-thiogalactosylpyranoside). Three variations of thisfusion protein were made, differing only in their cleavage site forliberating zFGF5 from MBP. One construct had a thrombin cleavage siteengineered between the MBP and zFGF5 domains. The second construct had aFactor Xa cleavage site, instead of a thrombin cleavage site. The thirdconstruct had an enterokinase cleavage site, instead of the thrombincleavage site.

[0194] The constructs were built as in-frame fusions with MBP inaccordance with the Multiple Cloning Site (MCS) of the pMAL-c2 vector(NEB), and according to the manufacturer's specifications. zFGF5 wasamplified via PCR using primers which introduced convenient cloningsites, as well as cleavage sites using the following oligonucleotideprimers: 1) for the thrombin construct: zc12652 (SEQ ID NO: 7) andzc12631 (SEQ ID NO: 8); 2) for the Factor Xa construct: zc15290 (SEQ IDNO: 9) and zc12631 (SEQ ID NO: 8); and 3) for the enterokinaseconstruct: zc15270 (SEQ ID NO: 10) and zc12631 (SEQ ID NO: 8). In eachcase, the native zFGF5 signal sequence was not amplified; the zFGF5 asexpressed begins at amino acid residue 26 of SEQ ID NO: 2 (Val waschanged to an Ala). The thrombin construct was built by inserting an XbaI-Sal I zFGF5 fragment into the Xba I-Sal I sites of pMAL-c2. The FactorXa construct was built by inserting a blunt-Sal I fragment into the XmnI-Sal I sites of the MCS. The enterokinase construct was built byinserting an Xba I-Sal I fragment into the Xba-Sal I sites of pMAL-c2.Once the constructs were built, they were transformed into a variety ofE. coli host strains and analyzed for high-level expression. Thethrombin construct (designated pSDH90.5) was transfected into DH1OBcells (GIBCO-BRL), while both the Factor Xa construct (designated pSDH117.3) and the enterokinase construct (designated pSDH116.3) weretransfected into TOP10 cells (Invitrogen, San Diego, Calif.). All threeMBP fusions are about 63 kD (43 kD in the MBP domain, and approximately20 kD in the zFGF5 domain).

[0195] B. Homologous Recombination/ zFGF5

[0196] Expression of zFGF5 in Pichia methanolica utilizes the expressionsystem described in co-assigned PCT publication WO97/17450, incorporatedherein by reference. An expression plasmid containing all or part of apolynucleotide encoding zFGF5 is constructed via homologousrecombination. The expression vector is built from pCZR204, whichcontains the AUGI promoter, followed by the αFpp leader sequence,followed by an amino-terminal peptide tag, a blunt-ended SmaIrestriction site, a carboxy-terminal peptide tag, a translational STOPcodon, followed by the AUG1 terminator, the ADE2 selectable marker, andfinally the AUG1 3′ untranslated region. Also included in this vectorare the URA3 and CEN-ARS sequences required for selection andreplication in S. cerevisisiae, and the AmpR and colEl ori sequencesrequired for selection and replication in E. coli. The zFGF5 sequenceinserted into this vector begins at residue 27 (Ala) of the zFGF aminoacid sequence.

[0197] To construct pSDHI 14, a plasmid for expression of zFGF5 in P.methanolica, the following DNA fragments were transformed into S.cerevisisae: 100 ng of the ‘acceptor vector’ pCZR204 that has beendigested with Smal; 1 μg of an XbaI-Sall restriction fragment liberatedfrom pSDH90.5, and encompassing zFGF5 coding sequence.; 1·μg of asynthetic, PCR-generated, double-stranded linker segment that spans 70base pairs of the aFpp coding sequence on one end and joins it to the 70base pairs of the amino-terminus coding sequence from the mature zFGF5sequence on the other was generated from the four oligonucleotideszc13497 (SEQ ID NO: 11); zc15131 (SEQ ID NO: 12); zc15132; (SEQ ID NO:18); zc15134 (SEQ ID NO: 13), of which the sense strand of a doublestranded sequence is shown in SEQ ID NO: 19 (5′ linker sequence (aFpp−>zFGF5 N-terminus)) and 1 μg of of a synthetic, PCR-generated,double-stranded linker segment that spans 70 base pairs ofcarboxy-terminus coding sequence from zFGF5 on one end with 70 basepairs of AUG1 terminator sequence was generated from the fouroligonucleotides 13529 (SEQ ID NO: 14); zc13525 (SEQ ID NO: 15) zc13526(SEQ ID NO: 16); zc13528 (SEQ ID NO: 17) of which the sense strand of adouble stranded sense is shown in the SEQ ID NO: 20 (3′ linker sequence(zFGF5 C-terminus ->AUGI terminator)). Ura+colonies were selected, andDNA from the resulting yeast colonies was extracted and transformnedinto E. coli. Individual clones harboring the correct expressionconstruct were identified by PCR screening with oligonucleotides zc13497(SEQ ID NO: 11) and zc13528 (SEQ ID NO: 12) followed by restrictiondigestion to verify the presence of the zFGF5 insert and DNA sequencingto confirm the desired DNA sequences had been enjoined with one another.Larger scale plasmid DNA is isolated for one of the correct clones, andthe DNA is digested with Sfi I to liberate the Pichia-zFGF5 expressioncassette from the vector backbone. The Sfi I-cut DNA is then transformedinto a Pichia methanolica expression host, designated PMAD16, and platedon ADE D plates for selection. A variety of clones are picked andscreened via Western blot for high-level zFGF5 expression.

[0198] More specifically, for small-scale protein production (e.g.,plate or shake flask production), P. methanolica transformants thatcarry an expression cassette comprising a methanol-regulated promoter(such as the AUG1 promoter) are grown in the presence of methanol andthe absence of interfering amounts of other carbon sources (e.g.,glucose). For small-scale experiments, including preliminary screeningof expression levels, transformants may be grown at 30° C. on solidmedia containing, for example, 20 g/L Bacto-agar (Difco), 6.7 g/L yeastnitrogen base without amino acids (Difco), 10 g/L methanol, 0.4 mg/Lbiotin, and 0.56 g/L of -Ade -Thr -Trp powder. Because methanol is avolatile carbon source it is readily lost on prolonged incubation. Acontinuous supply of methanol can be provided by placing a solution of50% methanol in water in the lids of inverted plates, whereby themethanol is transferred to the growing cells by evaporative transfer. Ingeneral, not more than 1 ml of methanol is used per 100-mm plate.Slightly larger scale experiments can be carried out using culturesgrown in shake flasks. In a typical procedure, cells are cultivated fortwo days on minimal methanol plates as disclosed above at 30° C., thencolonies are used to inoculate a small volume of minimal methanol media(6.7 g/L yeast nitrogen base without amino acids, 10 g/L methanol, 0.4mg/L biotin) at a cell density of about 1 x 10⁶ cells/ml. Cells aregrown at 30° C. Cells growing on methanol have a high oxygenrequirement, necessitating vigorous shaking during cultivation. Methanolis replenished daily (typically 1/100 volume of 50% methanol per day).

[0199] For production scale culturing, fresh cultures of high producerclones are prepared in shake flasks. The resulting cultures are thenused to inoculate culture medium in a fermenter. Typically, a 500 mlculture in YEPD grown at 30° C. for 1-2 days with vigorous agitation isused to inoculate a 5-liter fermenter. The cells are grown in a suitablemedium containing salts, glucose, biotin, and trace elements at 28° C.,pH 5.0, and >30% dissolved O₂. After the initial charge of glucose isconsumed (as indicated by a decrease in oxygen consumption), aglucose/methanol feed is delivered into the vessel to induce productionof the protein of interest. Because large-scale fermentation is carriedout under conditions of limiting carbon, the presence of glucose in thefeed does not repress the methanol-inducible promoter.

Example 10

[0200] Purification of zFGF5

[0201]E.coli fermentation medium was obtained from a strain expressingzFGF5 as a Maltose Binding protein fusion (pSDH90.5, as describedabove). The MBPzFGF5 fusion was solubilized during sonication or Frenchpress rupture, using a buffer containing 20 mM Hepes, 0.4 M Nacl, 0.01 MEDTA, 10 mM DTT, at pH 7.4. The extraction buffer also included 5 μg/mlquantities of Pepstatin, Leupeptin, Aprotinin, Bestatin. Phenyl methylsulfonylfluoride (PMSF) was also included at a final concentration of0.5 mM.

[0202] The extract was spun at 18,000×g for 30 minutes at 4° C. Theresulting supernatent was processed on an Amylose resin (Pharmacia LKBBiotechnology, Piscataway, N.J.) which binds the MBP domain of thefusion. Upon washing the column, the bound MBPzFGF5 fusion was eluted inthe same buffer as extraction buffer without DTT and protease inhibitorsbut containing 10 mM Maltose.

[0203] The eluted pool of MBPzFGF5 was treated with 1:100 (w/w) Bovinethrombin to MBPzFGF5 fusion. The cleavage reaction was allowed toproceed for 6 to 8 hours at room temperature, after which the reactionmixture was passed over a bed of Benzamidine sepharose (Pharmacia LKBBiotechnology, Piscataway, N.J.) to remove the thrombin, using the sameelution buffer as described above for Amylose affinity chromatography.

[0204] The passed fraction, containing the cleaved product zFGF5 andfree MBP domain were applied to a Toso Haas Heparin affinity matrix(Toso Haas, Montgomeryville, PA) equilibrated in 0.5 M NaCl, 20 mMHepes, 0.01 M EDTA at pH 7.4. The MBP and zFGF5 both bound to heparinunder these conditions. The bound proteins were eluted with a 2 to 3column volume gradient formed between 0.5M NaCl and 2.0 M NaCl in columnbuffer.

[0205] The MBP eluted early, at about 0.7 M NaCl, and the cleaved zFGF5eluted at about 1.3 M NaCl. The pooled zFGF5 fractions were passedthrough the amylose step once again to remove any residual MBPzFGF5 thatis a minor contaminant. The purified material was designated zFGF5-Hep2,and shows a single highly pure species at ˜20 kDa on reducing SDS-PAGEanalysis.

[0206] Amino acid N-terminal sequencing yielded the native N-Terminalsequence but Mass Spectrophotometry data revealed molecular massesindicating that the C-Terminus must be truncated at residue 196 (Lys) ofSEQ ID NO: 2, where a “dibasic site” is present. zFGF5 protein was verystable in 1.3 M NaCl. Upon dialysis into PBS, the zFGF5 aggregated andleft the solution phase. Therefore, formulations that include heparinand other “polyanions” may be used to prevent the aggregation of purezFGF5.

Example 1

[0207] Production of Antibodies

[0208] Antibodies for ZFGF5 were produced, using standard techniquesknown in the art and described previously, by immunizing guinea pigs,rabbits and mice with peptides QTRARDDVSRKQLRLYC (SEQ ID NO: 2 aminoacid residue 40 to residue 56), designated zFGF-1; YTTVTKRSRRIRPTHRAC(SEQ ID NO: 2 amino acid residue 191 to residue 207, with an additionalCys at the C-terminus), designated zFGF5 or the full-length zFGF5polypeptide as shown in SEQ ID NO: 2, plus the MPB fusion protein, anddesignated MBP-FGF5. Peptides were conjugated through Cys residues usingMaleimide-activated KLH (Pierce Chemical Co., Rockford, Ill.).

[0209] Table 7 is a description of the animals, immunization levels andantibody separations. TABLE 7 Peptide or Protein animal immun. level Abproduced ZFGF5-1 G.P.  50 ug/animal initial Affinity purified  25ug/animal boost and IgG fractionated. Rabbit 100 ug/animal initialAffinity purified  50 ug/animal boost and IgG fractionated. ZFGF5-2 G.P. 50 ug/animal initial Affinity purified  25 ug/animal boost and IgGfractionated. Rabbit 100 ug/animal initial Affinity purified.  50ug/animal boost and IgG fractionated. ZFGF5- Mouse  20 ug/animal initialMBP  10 ug/animal boost Rabbit 200 ug/animal initial Affinity purified100 ug/animal boost

Example 12

[0210] A. Effects of zFGF5 on ob/ob Mice

[0211] The effects of zFGF5 on adipocytes and fat metabolism wereexamined using female ob/ob mice (C57B1/6J, Jackson Labs, Bar Harbor,Me.). The mice are obese, insulin resistant and have “fatty bone”. Themice were weighed and all were found to be the same weight, and wereinjected IV with 1011 particles per mouse of AdCMVzFGF5 or either salineor Ad5CMV-GFP for controls, as described in Example 7. 17 days afterinjection, the control mice injected with Ad5CMV-GFP had gained5.342±0.5 grams of body weight compared to the day of injection, whilethe AdCMVzFGF5 treated mice lost 3.183±0.743 grams of body weight.

[0212] B. Effect of Pure zFGF5 Protein on Sprague-Dawley Rats

[0213] In addition, zFGF5 produced a dose dependent reduction in foodintake within 24 h of treatment. Individual male Sprague-Dawley ratswere given a single bolus IV injection of zFGF5 via an indwelling venouscatheter at dose levels of 8 mg/kg, 40 mg/kg and 76 mg/kg. Twoadditional animals were given vehicle alone. On day 14 post dose, theseanimals as well as two untreated male animals of the same age wereeuthanized by CO2, necropsied and tissues collected and preserved in 10%buffered formalin. Food intake was reduced 30% and 80% by the injectionof 9.0 and 40.0 mg/kg zFGFS, respectively. No further reduction in foodintake was seen with the 76 mg/kg dose. Food intake increased slowlyover the next ten days to near normal levels. The reduction in foodintake was associated with a drop in body weight in the zFGF5 treatedanimals that began with 24h and remained suppressed for 10 days in thehigh dose zFGF5 groups. The maximal drop in body weight over the courseof these experiments was 20-30% of the initial weight. A normal rate ofincrease in body weight was seen between days 10 and 14 in theseanimals.

[0214] These data suggest that zFGF5 will act as a satiety factor andwould be useful for treating obesity disorders.

Example 13

[0215] A. Cloning of Mouse zFGF5

[0216] A cDNA for the mouse ortholog of zFGF5 was isolated from a mouseembryo library. Oligonucleotide primers were designed from the fulllength human zFGF5 sequence (ZC17578 and ZC17579, SEQ ID NOS: 37 and 38,respectively). A PCR reaction was done using 2 μl of library as templateand ExTaq polymerase (PanVera, Madison, Wis.) under the followingconditions 1 cycle at 94° C. for 15 seconds; 35 cycles at 94° C. for 15seconds, 60° C. for 20 seconds, 72° C. for 30 seconds; and 1 cycle at72° C. for 10 minutes. The reaction mixture was incubated a 4° C.overnight. After the first reaction was screened, no positive cloneswere identified and the procedure was repeated until a positive clonewas identified. The positive clones were identified by transformingElectroMAZ DH1OB cells (GibcoBRL) with 1 μl of reaction mixture at 2.3kV. The cells were plated on culture plates containing ampicillin andmethicillin and incubated at room temperature for 3 days.

[0217] A DNA fragment obtained by PCR as described above wasradiolabeled using a Multiprime DNA Labeling System (Amersham) and usedas a probe for filters lifted from culture plates. The filter lifts werehybridized overnight at 65° C. in EXPRESS HYB (Clontech). Afterhybridiziation, the filters were washed in buffer of 0.25X SSC, 0.25%SDS, 1 mM EDTA at 65° C., 6 times.

[0218] Positive clones were identified and cDNA inserts were screened.The clones identified had truncations at the 5′ end, complete at the 3′ends and included 3′ UTR. One clone, designated LC 7-2 had the longest5′ end when compared to the human zFGF5 sequence. Sequence analysisverified that approximately 52 bp of 5′ sequence were missing and thatthis sequence was in the signal sequence and that the entire nucleotidesequence encoding the mature polypeptide was intact.

[0219] B. Northern Analysis

[0220] Northern analyses were performed using Mouse Multiple TissueBlots from Clontech (Palo Alto, Calif.), mouse heart blots (prepared atZymoGenetics, Inc.) and mouse dot blots (Clontech). Usingoligonucleotides ZC17579 (SEQ ID NO: 29) and ZC17578 (SEQ ID NO: 40) andthe mouse zFGF5 as a template, a probe was generated. The DNA probe wasradioactively labeled using a random priming MEGAPRIME DNA labelingsystem (Amersham, Arlington Heights, Ill.) according to themanufacturer's specifications. The probe was purified using a NUCTRAPpush column (Stratagene Cloning Systems, La Jolla, Calif.). EXPRESSHYB(Clontech, Palo Alto, Calif.) solution was used for prehybridization andas a hybridrizing solution for the Northern blots. Hybridization tookplace overnight at 68° C., and the blots were then washed in 2X SSC and0.05% SDS at RT, followed by a wash in 0.IX SSC and 0.1% SDS at 50° C.Multiple bands were observed at with predominate bands at approximately0.6-0.8 kb, 1.2 kb and 2.2-2.4 kb bands depending on the blot used.Signal intensity was highest for spleen with slightly lower intensitysignals in heart, lung, liver, skeletal muscle, kidney and testis. Mousedot blots with the same probe were positive only for spleen and day 17mouse embryo. Mouse heart mRNA northerns were probed and results werepositive for C57 Black, CD1, neonatal heart, and day 16 and day 20embryo, with strongest signal present in the day 16 embryo. BALB C mouseheart did not have a signal present.

[0221] Because the results in the mouse tissue did not directlycorrelate with results seen in the human tissue, a new probe wasdesigned. The new probe was designed specifically to exclude thepossibility that any members of the FGF family other than zFGF5 werepositive by Northern analysis. The probe was prepared using PCR witholigonucleotides ZC195687 (SEQ ID NO: 41) and ZC19633 (SEQ ID NO: 42)and template DNA from the mouse cDNA of zFGF5. The reactions wereessentially the same as described above. The mouse heart blot waspositive for C57 Black mouse, neonatal mouse, days 16 and 20 mouseembryo, with signals strongest in the neonatal heart and day 16 MRNA.The dot blots were positive for spleen and epididymus. It appeared thatthere was some variability for mouse mRNA expression, unlike humantissue, where heart mRNA consistently was the primarily tissue in whichzFGF5 was expressed in humans. Similar variability was seen with ratnorthern analysis.

Example 14

[0222] In vivo Study of Cardiomyopathic Rats

[0223] Rats infused subcutaneously with epinephrine for 2 weeks developa cardiomyopathy quite similar to human idiopathic dilatedcardiomyopathy (Deisher et al., Am. J. Cardiovasc. Pathol. 5(1):79-88,1994 and Deisher et al., J. Pharmacol. Exp. Ther. 266(1):262-269, 1993.)

[0224] The effect of zFGF5 on the initiation and progression of thecatecholamine-induced cardiomyopathy was evaluated by administeringzFGF5 by intra-pericardial injection to male, Sprague-Dawley ratsreceiving subcutaneous infusions of epinephrine or saline.

[0225] In one protocol, rats (300 gms) were implanted with subcutaneoussaline- or epinephrine-filled osmotic mini-pumps under light etheranesthesia. 96 hours following minipump implantation, a singleintra-pericardial injection of vehicle (n=25) or zFGF5 at 25, 250 or 500μg/kg was given (n=10 per dose). Mortality was monitored for anadditional two weeks, at the end of which the rats were sacrificed, thehearts were weighed wet, and fixed in 10% neutral buffered formalin forhistology.

[0226] The zFGF5 had no effect on mortality, body weight, heart weightor cardiac fibrosis in saline-infused rats.

[0227] In epinephrine-infused rats, the 25 μg/kg and 250 μg/kg dosesreduced mortality from 32% in vehicle injected rats to 0% in 25 μg/kgand 10% in 250 μg/kg injected rats. The highest zFGF5 dose, 500 Ag/kg,reduced mortality to 20% compared to vehicle injected rats, however thiswas not statistically significant. Cardiac fibrosis was determined byscoring Masson's Trichrome stained heart sections. Three sections werescored for each heart, and the average score taken. The fibrosis scorefor the vehicle-infused hearts was 1.26±0.25, while the score for the 25μg/kg zFGF5 injection was 1.74±0.23, the 250 Ag/kg injection was1.38±0.29, and the 500 μg/kg injection was 0.81±0.10. The dose of zFGF5which completely prevented mortality increased the cardiac fibrosisscore (25 μg/kg), while the dose which had no effect on mortalityreduced the cardiac fibrosis score (500 μg/kg). These results indicatethat a pro-fibrotic activity can be beneficial in the setting of heartfailure of varying etiologies, of which can include myocardial infarct(MI), idiopathic dilated cardiomyopathy (IDCM), hypertrophiccardiomyopathy, viral myocarditis, congenital abnormalities, andobstructive diseases.

[0228] In another protocol, the rats (300 gms) were anesthetized by anintra-muscular injection of an anesthetic cocktailketamine:rompun:acepromazine (1:1:0.1). Subcutaneous epinephrine-filledosmotic mini-pumps were implanted, and either vehicle or zFGF5 wasinjected intra-pericardially immediately afterward at 25 Ag/kg (n=25 pergroup). For the vehicle injected rats, 21% had died within 6 daysfollowing the epinephrine-filled minipump implantation, while none ofthe zFGF5 injected rats had died. By the end of the 2 week epinephrineinfusion period, 25% of the vehicle-injected rats had died, while only22% of the zFGF5-injected rats had died. In this model, zFGF5co-treatment at the time of minipump implantation delayed mortality byat least 7 days.

Example 15

[0229] Mammalian Expression Constructs

[0230] An expression plasmid containing all or part of a polynucleotideencoding zFGF5 is constructed via homologous recombination. A fragmentof zFGF5 cDNA is isolated using PCR that includes the polynucleotidesequence from nucleotide 1 to nucleotide 621 of SEQ ID NO: 1 or SEQ IDNO: 37, with flanking regions at the 5′ and 3′ ends corresponding to thevectors sequences flanking the zFGF5 insertion point. The primers forPCR each include from 5′ to 3′ end: 40 bp of flanking sequence from thevector and 17 bp corresponding to the amino and carboxyl termini fromthe open reading frame of zFGF5.

[0231] Ten μl of the 100 μl PCR reaction is run on a 0.8% LMP agarosegel (Seaplaque GTG) with 1×TBE buffer for analysis. The remaining 90 μlof PCR reaction is precipitated with the addition of 5 μl 1 M NaCl and250 μl of absolute ethanol. The plasmid pZMP6 which has been cut withSmal is used for recombination with the PCR fragment. Plasmid pZMP6 wasconstructed from pZP9 (deposited at the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, and isdesignated No. 98668) with the yeast genetic elements taken from pRS316(deposited at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, and designated No. 77145), an IRESelement from poliovirus, and the extracellular domain of CD8, truncatedat the carboxyl terminal end of the transmembrane domain. pZMP6 is amammalian expression vector containing an expression cassette having thecytomegalovirus immediate early promoter, immunoglobulin signal peptideintron, multiple restriction sites for insertion of coding sequences, astop codon and a human growth hormone terminator. The plasmid also hasan E. coli origin of replication, a mammalian selectable markerexpression unit having an SV40 promoter, enhancer and origin ofreplication, a DHFR gene, the SV40 terminator, as well as the URA3 andCEN-ARS sequences required for selection and replication in S.cerevisiae.

[0232] One hundred microliters of competent yeast cells (S. cerevisiae)are independently combined with 10 μl of the various DNA mixtures fromabove and transferred to a 0.2 cm electroporation cuvette. The yeast/DNAmixtures are electropulsed at 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol and the yeast is plated in two300 μl aliquots onto two URA-D plates and incubated at 30° C. Afterabout 48 hours, the Ura+yeast transformants from a single plate areresuspended in 1 ml H₂0 and spun briefly to pellet the yeast cells. Thecell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture is added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase is transferred to a fresh tube, and theDNA precipitated with 600 μl ethanol (EtOH), followed by centrifugationfor 10 minutes at 4° C. The DNA pellet is resuspended in 10 μl H2O.

[0233] Transformation of electrocompetent E. coli cells (DH10B,GibcoBRL) is done with 0.5-2 ml yeast DNA prep and 40 ul of DH1OB cells.The cells are electropulsed at 1.7 kV, 25 μF and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bacto′ Tryptone (Difco, Detroit, Miss.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCI2, 10 mMMgSO4, 20 mM glucose) is plated in 250 μl aliquots on four LB AMP plates(LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin).

[0234] Individual clones harboring the correct expression construct forzFGF5 are identified by restriction digest to verify the presence of thezFGF5 insert and to confirm that the various DNA sequences have beenjoined correctly to one another. The insert of positive clones aresubjected to sequence analysis. Larger scale plasmid DNA is isolatedusing the Qiagen Maxi kit (Qiagen) according to manufacturer'sinstruction.

Example 16

[0235] Mammalian Expression of zFGF5

[0236] CHO DG44 (Chasin et al., Som. Cell. Molec. Genet. 12:555-666,1986) are plated in 10 cm tissue culture dishes and allowed to grow toapproximately 50 to 70% confluency overnight at 37° C. , 5% CO₂, inHam's F12/FBS media (Ham's F12 medium, (Gibco BRL, Gaithersburg, Md.),5% fetal bovine serum (Hyclone, Logan, UT), 1% L-glutamine (JRHBiosciences, Lenexa, KS), 1% sodium pyruvate (Gibco BRL)). The cells arethen transfected with the plasmid zFGF5/pZMP6, using Lipofectamine™(Gibco BRL), in serum free (SF) media formulation (Ham's F12, 10 mg/mltransferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1%sodium pyruvate). ZFGF5/pZMP6 is diluted into 15 ml tubes to a totalfinal volume of 640 μl with SF media. 35 μl of Lipofectamine™ (GibcoBRL) is mixed with 605 μl of SF medium. The Lipofectamine™ mix is addedto the DNA mix and allowed to incubate approximately 30 minutes at roomtemperature. Five milliliters of SF media is added to theDNA:Lipofectamine™ mixture. The cells are rinsed once with 5 ml of SFmedia, aspirated, and the DNA:Lipofectamine™ mixture is added. The cellsare incubated at 37° C. for five hours, then 6.4 ml of Ham's F12/10%FBS, 1% PSN media is added to each plate. The plates are incubated at37° C. overnight and the DNA:Lipofectamine™ mixture is replaced withfresh 5% FBS/Ham's media the next day. On day 3 post-transfection, thecells are split into T-175 flasks in growth medium. On day 7postransfection the cells are stained with FITC-anti-CD8 monoclonalantibody (Pharmingen, San Diego) followed by anti-FITC-conjugatedmagnetic beads (Miltenyi Biotec, Auburn, Calif.). The CD8 positive cellsare separated by Miltenyi mini-MACS columns according to manufacturer'sdirections (Miltenyi Biotec), and put into DMEM/Ham's F12/5% FBS withoutnucleosides but with 50 nM methotrexate (selection medium).

[0237] Cells are plated for subcloning at a density of 0.5, 1 and 5cells per well in 96 well dishes in selection medium and allowed to growout for approximately two weeks. The wells are checked for evaporationof medium and brought back to 200 μl per well as necessary during thisprocess. When a large percentage of the colonies in the plate are nearconfluency, 100 μl of medium is collected from each well for analysis bydot blot, and the cells are fed with fresh selection medium. Thesupernatant is applied to a nitrocellulose filter in a dot blotapparatus, and the filter is treated at 100° C. in a vacuum oven todenature the protein. The filter is incubated in 625 mM tris glycine, pH9.1, 5mM βmercaptoethanol, at 65° C., 10 minutes, then in 2.5% non-fatdry milk Western A Buffer (0.25% gelatin, 50 mM TrisHCI pH 7.4, 150 mMNaCl, 5 mM EDTA, 0.05% Igepal, Sigma) overnight at 4° C. on a rotatingshaker. The filter is incubated with the antibody-HRP conjugate in 2.5%non-fat dry milk Western A buffer for 1 hour at room temperature on arotating shaker. The filter is washed three times at room temperature inPBS plus 0.01% Tween 20, 15 minutes per wash. The filter was developedwith ECL reagent according to manufacturer's directions (Amersham,Arlington Heights, ILL.), and exposed to film (Hyperfilm ECL, Amersham)approximately 5 minutes. Positive clones ate trypsinized from the 96well dish and transferred to 6 well dishes in selection medium forscaleup and analysis by Western blot.

Example 17

[0238] Expansion of Cells From Bone Marrow

[0239] Assays were performed to measure the frequency of fibroblastcolony forming units from monkey low density, non-adherent cellsisolated from bone marrow. This assay is indicative of mesenchymal stemcell frequency.

[0240] One half of a 96 well microtiter plate is inoculated with cellsat a density of 10,000 cells/well and the other half of the plate isinoculated with cells at a density of 1,000 cells/well. The culturemedium is αMEM (GIBCO-BRL, Gaithersburg, Minn.), 2% bovine serumalbumin, 10 μg/ml insulin, 200 μg/ml transferrin, antibiotic and 50 μM?-Mercaptoethanol. The cells are incubated at 37° C. in 5% CO₂ for 14days and then stained with toluidine blue to improve cell visibility andexamined microscopically. Positive wells have at least 50 cellsexhibiting a “stromal” morphology, i.e., large, spread out cells. Thepositive control is medium containing 20% fetal bovine serum. Resultsdemonstrated that zFGF5, at a concentration of 100 ng/ml increased thefrequency of CFU-F to levels equivalent to the positive control of 20%FBS.

Example 18

[0241] Effects of zFGF5 on Neural Cells

[0242] A. Neurite Outgrowth Assay-Relative Efficacy of zFGF5

[0243] The effect of zFGF5 on PC12, rat pheochromocytoma cells withneural potential (ATCC No. CRL-1721, American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209) examined using thefollowing growth factors, each at 3 dilutions:

[0244] 1 μg/ml, 100 ng/ml, and 10 ng/ml neural growth factor (NGF;source and location) in medium containing RPMI 1640 (R&D Systems,Minneapolis, Minn.)

[0245] 1 μg/ml, 100 ng/ml, and 10 ng/ml human basic FGF (R&D Systems,Minneapolis, Minn.)

[0246] 1 μg/ml, 100 ng/ml, and 10 ng/ml zFGF5 (recombinantly produced inE. coli .)

[0247] 1 μg/ml, 100 ng/ml, and 10 ng/ml zFGF5 (recombinantly produced inCHO cells)

[0248] The PC12 cells were plated at a concentration of 5×10⁴/ml ontocollagen coated 24 well culture plates and incubated for 48 hours in theappropriate medium. After 48 hours, the medium was changed to includeone of the cytokines described above and then changed again every 2days. The wells were scored for relative neurite outgrowth on days 6 and9.

[0249] Neurite outgrowth was induced with each of the cytokines. NGF andbFGF appeared to have similar affinity, while zFGF5 had significantlylower affinity. NGF exerted the greatest extent of neural outgrowthactivity, followed by bFGF, with significantly lower activity seen withzFGF5.A second experiment showed that zFGF5 has activity onneural-derived cells PC-12 cells .

[0250] B. Specificity and Efficacy of zFGF5 For Neural-derived Cells

[0251] Under a variety of culture conditions, in naive and primed cells,zFGF5 promoted neurite outgrowth. Survival of these cultures underserum-free conditions was also enhanced by the addition of zFGF5. ZFGF5protein was active over a concentration range of .03-1.0 ug/ml,suggesting that PC-12 cells contain a receptor that recognizes zFGF5.

[0252] To evaluate whether PC-12 cells express any of the known FGFreceptors, mRNA was isolated from cultures of PC-12 cells and the levelsof FGFR mRNA were measured by PCR using primers specific for FGFR3c. Aband of the correct size was observed only in the samples amplified withFGFR3c primers indicating that FGFR3c is a high affinity zFGF5 receptoron these cells.

[0253] The expression of zFGF5 mRNA was also examined in mouse braintissue by in situ hybridization with probes specific for zFGF5.Expression of zFGF5 mRNA was observed in neurons throughout the brain,expression was exceptionally high in Purkinje cells of the cerebellum,neurons in the cerebrum, thalamus, and hippocampus. Staining was alsoobserved in astrocytes throughout the brain. These results suggest thatzFGF5 plays a role in normal brain homeostasis.

[0254] These data suggest that zFGF5 will be useful for treating avariety of neurological conditions such as stroke, spinal cord injuries,dementia, and other neurological syndromes.

Example 19

[0255] Identification of a Target Cell

[0256] Identification of a putative mesenchymal stem cell as a targetfor zFGF5 was made using FITC-labeled protein and neonatal mouse hearttissue.

[0257] ZFGF5, purified as described above, was dialyzed into 0.1 Msodium bicarbonate pH 9.0. Fluorescein isothiocyanate (FITC; MolecularProbes, Eugene, OR) was dissolved at 1 mg/ml in the same buffer withoutexposure to strong light. The mixture was prepared containing 1 mgFITC/1 mg zFGF5, and reacted for 1-2 hours in the dark at roomtemperature. The reaction was stopped by adding 1 M glycine to a finalconcentration of 0.1 M, then reacted for 1 hour at room temperature. Themixture was then dialyzed against 0.1 M sodium biocarbonate to make a1:500-1:1000 dilution for 3 hours. The dialysis solution was changed andthe process repeated for 3-18 hours to remove unlabeled FITC.

[0258] Neonatal mouse heart ventricles were isolated, minced, andrepeatedly washed in phosphate buffered solution (PBS) until all redblood cells and debris were removed. The minced ventricles were placedin a solution containing 18 ml PBS and 1% glucose and 1 ml of 2%DNAse/Collagenase solution was added. The mixture was incubated on ashaker for 30 minutes at 37° C. The supernatant was discarded and theprocess was repeated once more. After incubation, the supernatant (˜20ml) was transferred to a tube containing 20 ml DF 20 (Dulbecco'sModified Eagle's Medium/Ham's Nutrient Mixture F12, 1:1 (GIBCO-BRL,Gaithersburg, Md.) and 20% fetal bovine serum). After mixing, the tubeswere centrifuged at 1650 rpms in a Beckman CS-6R centrifuge (Beckman,Fullerton, Calif.) at 4° C. for 10 minutes. The supernatant wasdiscarded and the pellet was resuspended in DF 10 (10% FBS). The cellswere kept cold and spun again and resuspended in 40 ml of DF 10. Thecell mixture was passed over a 40 μm filter (Becton Dickinson, Detroit,MI) and counted using a hemacytometer.

[0259] The cells were incubated in FITC-labeled zFGF5 at 4° C. for 30minutes at a concentration of 2×10⁶ cells/1 μg zFGF5. After incubation,the cells were spun at 1650 rpms in a Beckman CS-6R centrifuge (Beckman)for 5 minutes. The supernatant was discarded and the pellet washed oncein 10 ml of DF 10 and resuspended in 4 ml DF 10.

[0260]10 μl of MACS anti-FITC microbeads (Miltenyi Biotech, Auburn,Calif.) were mixed with 10⁷ cells in 4 ml of DF10 and incubated at 4° C.for 30 minutes.

[0261] MACS positive selection type LS+ separation columns (MiltenyiBiotech) were washed with 3 ml of MAC buffer (PBS, 0.5% BSA, 2 mM EDTA)and the cell/bead mixture was washed in 10 ml MAC buffer and thenresuspended in 6 ml MAC buffer. The cell/bead mixture was dividedbetween the two columns and the first negative fraction was discarded.1.5 ml of 0.6 M NaCl was added to each column and eluted but notcollected. The columns were then washed with 1.5 ml MAC buffer. Thecells bound with FITC-labeled zFGF5 were collected by adding 3 ml MACbuffer, removing the column from the magnet and flushing out thepositive cells using the plunger. The positive cell fraction was platedin a T75 flask and 50 ml of plating medium was added (DF with 15% FBSand antibiotics). The cells were incubated at 37° C. for 1 week andcounted. The yield of positive cells was approximately 0.1% of originaltotal cells counted.

[0262] Cells binding FITC-labeled zFGF5 were examined by transmissionelectronmicroscopy (TEM). The cells were between 3-5 microns indiameter. The cell nuclei occupied the majority of the cell volume, andfew cytoplasmic organelles were apparent. The phenotype identified byTEM identifies the zFGF5-isolated cells as primitive mesenchymal stemcells.

Example 20

[0263] Identification of an FGF Receptor With Specificity For zFGF5

[0264] A panel of six BAF 3 cell lines were transfected to express eachof the known FGF receptor splice variants (FGFR1α-IIIb,-IIIc,FGFR2α-IIIb,-IIIc, FGFR3α-IIIb, IIIc). The transfected cell lines wereused to assess the receptor specificity of zFGFS. BAF3 cells do notexpress their own FGF receptors and are dependent on IL-3 for growth.Wild type BAF3 cells, thus, do not respond to FGF, and removal of IL-3from the growth media abolishes cell proliferation. In transfected celllines the addition of 2.0 ug heparin/ml and an FGF that activates aspecific FGF receptor stimulates cell proliferation. Each cell line wastested in mitogenesis assays using zFGF5.

[0265] These data were confirmed and extended using FGFR3-IIIcexpressing cells and soluble FGFRs. The soluble FGFRs contained theligand binding domain of each FGF receptor fused to the Fc portion ofhuman immunoglobulin G. In competition studies using zFGF5 to stimulatecell proliferation, only FGFR3α-IIIc-Fc and FGFR4-Fc produced a highaffinity blockade of cell growth. Some inhibition was also observed withFGFR2α-IIIc-Fc but the affinity was about 100-fold lower. Table 8illustrates the effects of zFGF5 on cell proliferation of FGFreceptor-expressing cells. TABLE 8 zFGF5 FGFR1 FGFR2 FGFR3 Concentration-IIIb -IIIc -IIIb -IIIc -IIIb -IIIc (ng/ml) Alamar Blue Units 1.6 0 2.60.2 241 0 1402 3.1 1.7 4.2 0.06 480 0 2003 6.2 1.3 14.2 0 715 0 258112.5 0 29.9 0.4 977 0 3201 25 10.9 86 0.2 1307 1.4 3764 50 0 224 0.51523 19 3958 100 0 488 1.3 1723 189 4117 200 0 912 1.3 1794 472 4205

[0266] The only high affinity response (EC50˜1-2 ng/ml) was seen withBAF3 cells expressing FGFR3a Mc. A lower affinity response (EC50˜10-20ng/ml) of activation was observed in cells expressing FGFR2α-IIIc.Activation of the 3c and 2c cells by zFGF5 was, moreover, completelydependent on exogenous heparin. Addition of zFGF5 did not enhanceproliferation of any of the FGFR “b” splice variant expressing cells.

[0267] To further explore the receptor selectivity of zFGF5, the abilityof a series of FGF receptor-human Fc fusion proteins (soluble receptors)was tested for their ability to compete for zFGF5-mediated cellproliferation in BAF3 cells expressing the FGFR3α-IIIc receptor. Of thesoluble receptors tested, only the FGFR4/Fc and FGFR3α-IIIc/Fc receptorsexhibited a high affinity competition for zFGF5-mediated cellproliferation. The FGFR2α-IIIc/Fc receptor did compete for zFGF5binding, however, the EC50 for this effect was more than 100-fold higherthan that seen with FGFR4/Fc and FGFR3α-IIIc/Fc. Competition was notobserved using the FGFR1α-IIIc/Fc or the FGFR2α-IIIb/Fc receptors. Table9 illustrates the inhibition of zFGF-5-mediated cell proliferation byFGF receptor-Fc Fusion proteins. TABLE 9 FGFR1 FGFR2 FGFR3 ConcentrationIIIc-Fc IIIb-Fc IIIc-Fc IIIc-Fc FGFR4 (fold-excess) Cell Proliferation(percent of control) 0 100 100 100 100 100 0.8 85 79 1.6 66 64 3.2 37 276..3 5 8 7.5 92 96 12.5 1 2 15.4 96 93 25 .2 1 31 99 91 50 0 0 62.5 104101 77 100 0 0 125 101 90 61 250 100 91 39 500 102 101 22 1000 92.3 99 6

[0268] Table 10 is a comparison of the receptor specificity of FGF-1,FGF-2, and zFGF-5. TABLE 10 FGF receptor FGF-1 FGF-2 zFGF5 1b + + −1c + + − 2b + − − 2c + + ± 3b + − − 3c + + + 4 + + +

[0269] These data demonstrate that of the known FGF receptors, zFGF5appears to preferentially bind to FGFR4 and FGFR3-IIIc. A comparison ofFGF receptor specificity of FGF-1, FGF-2, and zFGF5, suggests that thereceptor binding specificity of zFGF5 is considerably more restrictedthan either FGF-1 or -2.

Example 21

[0270] Stimulation of Chrondocytes and Cartilage Matrix

[0271] Adenovirus expressing zFGF5 was injected into the external eartissue of nude mice. Compared to infection with the control “empty”adenovirus, ears infected with adenovirus expressing zFGF5 were visiblythicker . Histologic analysis revealed that the increase in thicknesswas due in part to the proliferation of chondrocyte cells within the earcartilage. Cells within this region stained positive for PCNA, a markerof cell proliferation, and were stained by both Alcian blue andToluidine blue, markers of collagen synthesis. There was little or nostaining with PCNA in contrast, in the chondrocyte zone of mouse earsinfected with the control adenoviral vector. These data suggested thatzFGF5 is a mitogen for chondrocytes.

[0272] To test this directly, chondrocytes were isolated from porcinearticular cartilage of the knee joint and cultured in the presence andabsence of zFGF5 protein. Proliferation of these cells, measured by[³H]thymidine incorporation was enhanced more than three-fold byincubation with zFGF5 over a concentration range of 10-500 ng/ml. Inaddition, the synthesis of glycosaminoglycans, measured by ³⁵Sincorporation which are characteristic of cartilage tissue, was enhancedin these cultures more than 4-fold by the addition of zFGF5 over asimilar concentration range. Table 11 illustrates the effects of zFGF5on chondrocyte proliferation and glycosaminoglycan production. TABLE 11zFGF5 [³H]Thymidine [³⁵S]Sulfate concentration incorporationincorporation (ng/ml) cpm cpm 0 35805 4834 10 43777 4242 50 65519 100100896 16457 200 107174 18612 500 125294

[0273] To assess whether zFGF5 mRNA is expressed in normal humantracheal and articular cartilage, probes specific for zFGFS mRNA wereprepared and zFGF5 mRNA levels were assessed by in situ hydridizationtechniques. These analyses revealed intense staining for zFGF5 mRNA inthe cytoplasm of mature chondrocytes in both tissues. No expression wasdetected in immature chondrocytes on the same section. The resultsdemonstrate that zFGF5 can stimulate the proliferation of maturechondrocytes in vivo and in vitro, can stimulate matrix production bythese cells, and is expressed by chondrocytes in human cartilage.

[0274] Further analysis of the role of zFGF5 in cartilage biology innormal and diseased states examined expression of mRNA for zFGF5 andFGFR3α-IIIc in human chondrosarcoma tissue. Expression of zFGF5 andFGFR3α-IIIc mRNAs was observed in chondrocytes throughout the tumortissue sections suggesting that zFGF5 may regulate cell proliferation inthese tumors.

[0275] Further confirmation of zFGF5-induced proliferation ofmesenchymal-derived tumor cells, in particular osteoblast- andchondrocyte-derived tumors, was shown by conjugating zFGF5 to the celltoxin saporin. Conjugates of cytotoxic polypeptides and zFGF5 componentswere prepared using standard techniques for conjugating polypeptides,and are described, for example, by Lappi et al., Biochem. Biophys. Res.Commun. 160:917 (1989), Soria et al., Targeted Diagn. Ther. 7:193(1992), Buechler et al., Eur. J. Biochem. 234:706 (1995), Behar-Cohen etal., Invest. Ophthalmol. Vis. Sci. 36:2434 (1995), Lappi and Baird, U.S.Pat. No. 5,191,067, Calabresi et al., U.S. Pat. No. 5,478,804, and Lappiand Baird, U.S. Pat. No.; 5,576,288. Additional approaches toconjugating polypeptide are known to those of skill in the art. Forexample, Lam and Kelleher, U.S. Pat. No. 5,055,291, describe theproduction of antibodies conjugated with either diphtheria toxinfragment A or ricin toxin.

[0276] When human osteosarcoma cells were incubated with a zFGF5-saporinconjugate and cell viability was determined, zFGF5-saporin conjugateproduced a dose-dependent killing of the cells with an EC50 of about 1.0nM. Saporin alone or other saporin conjugated to several unrelatedproteins were without effect, suggesting that zFGF5 conjugated to acytotoxin has specificity for mesenchymal-derived tumor cells and may beused to inhibit such tumors.

Example 22

[0277] zFGF5 Affects Wound Healing

[0278] Investigation of whether zFGF5 would promote wound healing wasmade using bFGF for comparison and PDGF as a positive control in male,19 weeks old, db/db mice. In the study, the mice received a dorsalfull-thickness skin excision of approximately lcm square. The wound wascovered with a semi-occlusive, self-adhesive dressing. The wounds weretreated topically, daily, with one of the following: vehicle, PDGF, bFGFor zFGF5. The treatments, 10 μg of a growth factor in 0.ml of PBS, wereinjected through the dressing onto the wound bed daily for 7 treatments.The mice were sacrificed on Day 18 and the whole wound bed plussurrounding normal skin were harvested for histology.

[0279] Wound closure was measured on Days 6, 10 and 18 by tracing theedge of the scar (the originally cut edge) and the advancing edge of thenew epithelium onto acetate transparencies. The areas defined by the twoedge tracings were measured and calculations of % Closure, %Re-epithelialization and % Contraction compared to Day 0 were done atthe three timepoints.

[0280] Wound fluid accumulated over the healing wound beds was removedand measured daily as another index of the rate of wound healing. Thevolume of wound fluid collected from the bFGF treated wounds was clearlygreater than any other group. This is consistent with the observationthat the bFGF treated wound beds appeared much more highly vascularizedthan the other 3 groups. The bFGF wounds were very bright red incomparison with the other groups. The open and re-epithelialized areasof the wounds were measured using the planimetry program, Optimas. Thestatistical analyses were done using the program InStat.

[0281] On Day (-2), the mice had the hair clipped from their dorsum andwere then depilated. On Day 0, the mice were anesthetized with Metofaneanesthetic; the denuded dorsum wiped with Povidone Iodine then 70%Isopropyl alcohol, and the corners of a lcm square template were markedand the skin within the defined area excised (approximately) underaseptic conditions. The mice were administered through the dressingtopically onto the wound bed daily for 7 treatments, startingimmediately following surgery. On Days 6, 10, 14 and 18 the wounds weretraced. Group 1 vehicle n = 8 Group 2 PDGF 10 μg n = 8 Group 3 bFGF5 10μg n = 8 Group 4 zFGF5 10 μg n = 8

[0282] Wound healing occurs by a combination of contraction andre-epithelialization. Percent Closure is the union of the %Re-epithelialization and % Contraction. In this study, the % Contractionwas equivalent in the groups treated with bFGF and PDGF. The grouptreated with zFGF5 had only slightly higher % Contraction than thevehicle treated group on Day 18. The earlier timepoints showedequivalent % Contraction between vehicle and zFGF5 groups.

[0283] There was a statistically significant increase in % Closurecompared to the vehicle treated wounds in the zFGF5-, FGF-2-, andPDGF-treated wounds on Day 18. Similar changes were observed for eachgroup for the percent re-epithelialization parameter. The changesobserved in the zFGF5-treated animals did not quite achieve statisticalsignificance.

[0284] Wound fluid accumulates in the wound from vessels that arerendered “leaky” by cytokines locally released from various cell types,such as histamine released from infiltrating macrophages. Thecomparatively “hyper-vascular” appearance of the wounds in the bFGFgroup is consistent with more extravasation of fluid in that group thanany other group. The zFGF5 group had more fluid accumulation than thevehicle group, but significantly less than the PDGF group, which wasintermediate in accumulated fluid volume.

[0285] The volumes of wound fluid collected were clearly differentbetween the groups. A table of the total volume collected per treatmentgroup per day follows. TABLE 12 Day Day Day Day Day Day Day Day 4 5 6 78 9 10 11 wound fluid accumulations: ml/day Vehicle 0 0 0 0.06 0.02 0 00 PDGF 0.1 0.29 0.35 0.5 0.42 0 0 0 FGF-2 0.05 0.72 1.04 0.81 0.54 0.050.45 0.01 zFGF5 0 0 0.12 0.1 0.07 0 0 0

[0286] The wounds treated with bFGF, however, looked extremely red andalso accumulated much more fluid than the other three groups. Grossly,the wounds had a “hyper-vascularized” and inflamed appearance.Histologic evaluation will reveal whether they actually contained moreinfiltrating white blood cells than the other groups.

Example 23

[0287] Reduction in Cerebral Infarct Volume in Mouse Stroke Model

[0288] Tests for the effects of zFGF5 on mice were done using the middlecerebral artery occlusion (MCAO) model of stroke. C57BL6 rmice weresubjected to cerebral ischemia by a 1.0 h occlusion of the anteriorcommunicating and middle cerebral arteries, followed by 24h ofreperfusion. Administration of vehicle or zFGF5 was by one of tworoutes: intracerebrovascular (ICV) injection (0, 0.05, 0.5, or 5.0 ugper mouse) given 2h prior to the onset of ischemia, or by iv infusion(0, 50, 100, 200 ug/kg for 2h) starting 15 min after the onset ofischemia.

[0289] Behavioral analysis was performed on each mouse 24 h after theonset of ischemia, the animals were sacrificed after the 24h behavioraltest, and infarct volume was measured by TTC staining (Koketsu et al.,Ann. Neurol. 35:451-457, 1994). Infarct volume in mm³ was measured bycomputer-assisted videodensitometry in seven sections taken rostral tocaudal through the infarct. Neurological deficits were graded from none(0) to most severe (3) as follows: 0, no observable deficit; 1, failureto extend right forepaw upon lifting of tail; 2, circling to thecontralateral side; 3, leaning to the contralateral side at rest or nospontaneous motor activity.

[0290] For each animal, thermistor probes were inserted into the rectumand temporalis muscle to monitor body and brain temperatures wasmaintained at 37° C. Mean arterial blood pressure and arterial blood pH,pO2, pCO2 were analyzed 10 min before occlusion, 10 min after occlusionand 10 min after reperfusion. Regional cerebral blood flow was measuredby laser-Doppler flowmetry with a fiber-optic probe inserted 2mmposterior, 6 mm lateral to the bregma on the ipsilateral hemisphere, thesite supplied by the proximal segment of the middle cerebral artery(MCA). Table 13 illustrates the effect of IV infusion of zFGF5 oninfarct volume. Table 14 illustrates the occlusion of the MCA reducedregional blood flow in all groups. TABLE 13 zFGF5 dose Infarct Volume ±SD μg/kg/hour mm³ 0 88 ± 25 50 63 ± 31 100 52 ± 22* 200 39 ± 17**

[0291] TABLE 14 Cerebral blood flow (% of control) zFGF5 dose beforeduring during (ug/kg/h) ischemia isehemia reperfusion 0 100 19 96 50 10021 97 100 100 22 95 200 100 24 98

[0292] Administration of zFGF5 by ICV injection 2.Oh prior to the onsetof cerebral ischemia or by iv infusion starting 15 min after ischemia,produced concentration-dependent reductions in infarct volume. Comparedto injection of vehicle, infarct volume was reduced more than 50% bytreatment with zFGF5 (p<0.003, n=8 mice for each experimentalcondition). In addition to the reduction in infarct volume, zFGF5appeared to reduce ischemia-induced neurological deficits.

[0293] There were no significant changes in mean arterial bloodpressure, P⁰2, pCO₂, or pH in any of the groups under any of theconditions tested. Compared with cerebral blood flow prior to ischemia,regional blood flow during ischemia was reduced about 75% confirmingthat cerebral blood flow was reduced under these conditions.

Example 23

[0294] Immunocytochemistry of zFGF5

[0295] Immunocytochemistry to detect zFGF5 protein was carried out onhuman brain tissue. The subjects and tissues used were the temporalcortex of a 70-year old male and an 83 year-old female, the cerebrum ofa 47 year-old female and brain tissue (no region specified) from a 71year-old male. All subjects died of cerebrovascular accidents. In theuninvolved areas of the grey matter, capillaries stained positive forzFGF5, but neurons were negative for staining. In areas of old infarct,capillaries, vascular endothelium, and macrophages stained positive forzFGF5 protein. Both the astrocytes surrounding the capillaries as wellas the endothelium were positively stained. Occasional microglial cellswere seen within the white matter that stained positive, as well assubpial glial cells. Regions adjacent to the infarcted area showedincreased staining of the capillary endothelium, astrocytes, andmacrophages.

[0296] Normal cerebral cortical tissue from a 55 year-old female whodied of an aortic aneurysm was also examined for zFGF5 immunoreactivity.Sections of grey matter showed neurons and astrocytes that were largelynegative for staining with antibody. Sections of white matter showedoligodendrocytes and astrocytes that were negative for staining. Smoothmuscle of cerebral vessels and capillaries, in contrast, were stronglypositive for staining within both the grey and white matter.

[0297] Thus, in cerebrovascular accident cases, staining for zFGF5 wasseen within vessels adjacent to the infarcted area, with decreasedstaining of the infarct itself. Staining was increased in areas ofhealing particularly within astrocytes surrounding capillaries and thecapillary endothelium itself. Macrophages and microglial cells were alsopositively stained within the infarct and the adjacent areas. Neurons inthese regions were largely negative for staining. In normal cerebralcortical tissue, smooth muscle of cerebral vessels and capillaries werestrongly positive.

[0298] The pattern of detection of zFGF5 protein and mRNA within thebrain suggests that zFGF5 might play a role in control of brainmicrovasuclature. In addition, zFGF5 may regulate communication betweenneuronal and non-neuronal cell types within this tissue, or may beupregulated in specific cell types, such as astrocytes and macrophages,in response to injury. The apparent differences in cellular localizationbetween zFGF5 protein and zFGF5 mRNA suggests either that zFGF5 istransported from its site of synthesis to depot sites within thevicinity or that significant species differences exist in the expressionof the zFGF5 gene in the central nervous system.

Example 24

[0299] Cartilage Repair in Goats

[0300] The effect of zFGF5 delivered in a slow degrading polymer matrixon healing of a cartilage defect is preformed using a goat cartilagedefect model. The experiment will demonstrate whether the polymer matrixalso acts as a scaffold for cellular and matrix ingrowth. The dosage ofzFGF5 is 0, 0.5, 5, 50, or 500 ng/mm³ defect volume. Efficacy of thecombined factor and slow release polymer is assessed using gross andhistological examination of the repaired tissue in the cartilage defectsite. The following doses are administered: Atrix Slow Release Polymerwith Vehicle (0.0 μgram zFGF5) n = 3 Atrix Slow Release Polymer with 0.3μgram zFGF5 n = 3 Atrix Slow Release Polymer with 3.0 μgram zFGF5 n = 3Atrix Slow Release Polymer with 30.0 μgram zFGF5 n = 3 Atrix SlowRelease Polymer with 300.0 μtgram zFGF5 n = 3

[0301] The control and test materials are supplied as a sterile packagedimplant containing the polymer and either vehicle or premixed solutioncontaining the appropriate zFGF5 concentration.

[0302] A total of 15 skeletally mature female Spanish goats will be usedfor this study. They will be acquired from an approved, USDA source. Thegoats will weigh between 50-80 lbs. at the start of the study. Thecontralateral joint from all animals are not be operated on and servefor baseline normal measurements.

[0303] Animal housing conditions conform with applicable laws andregulations relating to laboratory animals, i.e., Animal Welfare Act,Public Law 89-544 as amended in Public Law 99-198, Federal Register52:16, United States Department of Agriculture—Animal and PlantInspection Service (USDA-APHIS), 1985 and Public Health Service Policyon Humane Care of Laboratory Animals, Office for Protection AgainstResearch Risks/National Institutes of Health (OPRR/NIH), September,1986. The animals are examined for any evidence of disease or lameness,and are Q-Fever negative, T/B negative, Brucellosis negative, CAEnegative.

[0304] The goats are maintained in large indoor runs (pens) or inoutdoor runs for several days following surgery, and when appropriate,the goats are moved to a large outdoor (fenced) paddock area for theremainder of the study. The goats have unrestricted activity at alltimes. The goats are fed a mixture of baled grass hay and alfalfa cubes.Tap water is provided ad libitum. However, feed is withheld 24 hoursprior to surgery.

[0305] Bodyweight measurements are taken from all animals once duringthe quarantine period, prior to surgery (Day 0) and at the end of thestudy (Day 56). Blood (approximately 5 ml) is taken from all animalsprior to surgery (Day 0) and at the end of the study (Day 56). The serumis harvested, placed in appropriately labeled screw top freezer tubes,and stored at −20° C.

[0306] The basic surgical procedure is identical for all subjects. Allsurgeries are performed under strict asepsis. Peri-operative antibioticsare dosed IM at 2.4 million units of Bicillin® at the beginning of theprocedure. Anesthesia is induced with a mixture of ketamine-xylazine,the subject intubated, and anesthesia maintained with a gaseous mixtureof Isoflurane and oxygen.

[0307] A lateral or medial trochlear defect is created in the rightfemoral condyle. The defects are approximately 6 mm in diameter andapproximately 2.5 mm in depth, and pass into the subchondral bone. Thedefects are made on either the lateral or medial wall of the distaltrochlear sulcus dependent on individual anatomy. Following creation ofthe defect, the wall may be undercut to provide for a mechanicalinterlock with the polymer matrix. Each defect is then filled with theappropriate test article.

[0308] The surgical approach consists of a curved, lateral skin incisionmade from the distal one-third of the left femur to the level of thetibial plateau and across to the medial side of the tibial spine. Usingthis method, the skin is bluntly dissected and retracted to allow alateral parapatellar approach into the stifle joint. An incision is madeparallel to the lateral border of the patella and patellar ligament.This extends from the lateral side of the fascia lata along the cranialborder of the biceps femoris and into the lateral fascia of the stiflejoint. The biceps femoris and attached lateral fascia are retractedallowing an incision into the joint capsule. The joint is extended andthe patella luxated medially exposing the stifle joint.

[0309] With the knee joint fully flexed, the appropriate location forthe point of drilling the defect on the trochlear sulcus is identifiedand marked with a surgical marker. A specially designed cartilage cutteris used to slice through the cartilage outer layer and prevent tearingof the cartilage. The approximate 6 mm diameter core cutter is usedunder power to create a fixed depth of approximately 2.5 mm, maintaininga plane perpendicular to the tangent of the sulcus. The core ofsubchondral bone and cartilage is carefully removed.

[0310] The cartilage defect is copiously flushed with sterile saline andthen dried prior to insertion of the test article. The polymer materialswill set-up in situ once it comes into contact with fluid. Theappropriate test material is then injected into the defect until thesurface is flush with the surrounding cartilage. Buffer is flooded overthe surface of the polymer and the polymer is allowed to set for 5minutes prior to carefully flushing the joint. The patella is thenreduced and the joint moved through a complete range-of-motion to ensurethat there is no impingement due to the implant. This is followed byroutine closure of the joint in three or four layers using appropriatesuture material. Post operative checks are made for any animaldisplaying signs of post operative discomfort. Post operative analgesicswill be given if the animals display any signs of distress ofdiscomfort. All treatments will be recorded in the appropriate studydocumentation.

[0311] All remaining animals are humanely sacrificed at Day 56 (8 weeks)postoperatively. Bodyweights are recorded immediately prior tosacrifice. Gross evaluation is performed on the heart, lungs, liver,spleen, kidneys, and popliteal and superficial inguinal lymph nodes forsigns of any systemic toxicity from the implant material. Lymph nodes inclose proximity to the joint are examined. The articulating surfacesopposing the defect sites are examined for any abnormal joint surface.Additionally, gross evaluations of the knee joints are made to determinethe cartilage repair based on previous scoring criteria listed in Table15. Femora, patellae, synovium, and popliteal lymph nodes are harvestedand placed into appropriately labeled containers. Immediately followingtissue harvest, gross morphological examination of the cartilage surfaceis done and photographic records made of each specimen.

[0312] After collection of the knee joints, the joints will be opened,photographed and the surface of the defect site scored as indicated inTable 15 and with standardized grading sheets. The synovial membranewill be examined for any inflammation. TABLE 15 Scoring Criteria forGross Morphological Evaluations characteristic grading score EdgeIntegration Full 2 (new tissue relative to native cartilage) Partial 1None 0 Smoothness of the cartilage surface Smooth 2 Intermediate 1 Rough0 Cartilage surface, degree of filling Flush 2 Slight depression 1Depressed/overgrown 0 Color of cartilage, opacity or Transparent 2translucency of the neocartilage Translucent 1 Opaque 0

[0313] Immediately after dissection and following gross joint surfaceobservations, the joints are placed in 10% phosphate buffered formalin(at least ten-fold volume) for at least 48 hours and histologicallyprocessed. After fixation in 10% phosphate buffered formalin, thespecimens are grossly trimmed to remove extra tissue. The tissue blocksare cut approximately ⅓ of the distance in from the exteriorimplant/tissue interface in order to examine them grossly. Contactradiographs are taken prior to the commencement of decalcification.

[0314] The tissues are decalcified in 10% EDTA until radiographs of thedecalcified sections assures complete decalcification. Once completedecalcification is determined, the specimens are dehydrated through anethanol series and paraffin embedded. The specimens are sectioned to5-10 μm. One section is stained with H&E and another sequential sectionwith Safranin 0 counter-stained with Fast Green. For histologic analysisof the sections, the scoring scale shown in Table 16 is used. TABLE 16characteristic grading score I. Nature of predominant hyaline cartilage4 tissue mostly hyaline cartilage 3 mixed hyaline and fibrocartilage 2mostly fibrocartilage 1 some fibrocartilage, mostly 0 nonchondrocyticcells II. Structural smooth and intact 3 characteristics superficialhorizontal lamination 2 A. Surface regularity fissures 1 severedisruption, including 0 fibrillation B. Structural integrity normal 2slight disruption, including cysts 1 severe disintegration 0 C.Thickness 100% of normal adjacent cartilage 2 50-100% of normalcartilage 1 0-50% of normal cartilage 0 D. Bonding to adjacent Bonded atboth ends of graft 2 cartilage Bonded at one end or partially at 1 bothends Not bonded 0 III. Freedom from cellular normal cellularity 2changes of slight hypocellularity 1 degeneration moderatehypocellularity or 0 A. Hypocellularity hypercellularity B. ChondrocyteNo clusters 2 clustering <25% of the cells 1 25-100% of the cells 0 IV.Freedom from Normal cellularity, no clusters, 3 degenerative changesnormal staining in adjacent cartilage Normal cellularity, mild clusters,2 moderate staining Mild or moderate hypocellularity, 1 slight stainingSevere hypocellularity, poor or no 0 staining V. A. Reconstitution ofnormal 3 subchondral bone reduced 2 minimal 1 none 0 B. Inflammatorynone/mild 2 response in moderate 1 subchondral bone severe 0 VI.Safranin-O staining normal 3 moderate 2 slight 1 none 0

[0315] Histologic Scoring Scale is modified for scoring the “Nature ofpredominant tissue”, Section I of the scale. In the 16 week goat study,if the tissue is scored as “4=hyaline cartilage” it essentially consistsof only hyaline cartilage, no trace of fibrocartilage. Scoring thenature of the repair tissue as “3=mostly hyaline cartilage” is given tosections which have some trace of fibrocartilage, but less than 25% asdetermined visually. A score of “2=mixed hyaline and fibrocartilage” isgiven to repair tissue which has both hyaline and fibrous tissue,varying from approximately 75% hyaline/25% fibrous to 25% hyaline/75%fibrous. A score of “1=mostly fibrocartilage” is given to repair tissuewhich show some traces (less than 25%) of hyaline, but is primarilyfibrous in nature. A score of “O=some fibrocartilage, mostlynon-chondrocytic” is given to repair tissue which does not exhibit anyhyaline tissue at all.

[0316] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 43 1 917 DNA Homo sapiens CDS (1)...(621) 1 atg tat tca gcg ccc tccgcc tgc act tgc ctg tgt tta cac ttc ctg 48 Met Tyr Ser Ala Pro Ser AlaCys Thr Cys Leu Cys Leu His Phe Leu 1 5 10 15 ctg ctg tgc ttc cag gtacag gtg ctg gtt gcc gag gag aac gtg gac 96 Leu Leu Cys Phe Gln Val GlnVal Leu Val Ala Glu Glu Asn Val Asp 20 25 30 ttc cgc atc cac gtg gag aaccag acg cgg gct cgg gac gat gtg agc 144 Phe Arg Ile His Val Glu Asn GlnThr Arg Ala Arg Asp Asp Val Ser 35 40 45 cgt aag cag ctg cgg ctg tac cagctc tac agc cgg acc agt ggg aaa 192 Arg Lys Gln Leu Arg Leu Tyr Gln LeuTyr Ser Arg Thr Ser Gly Lys 50 55 60 cac atc cag gtc ctg ggc cgc agg atcagt gcc cgc ggc gag gat ggg 240 His Ile Gln Val Leu Gly Arg Arg Ile SerAla Arg Gly Glu Asp Gly 65 70 75 80 gac aag tat gcc cag ctc cta gtg gagaca gac acc ttc ggt agt caa 288 Asp Lys Tyr Ala Gln Leu Leu Val Glu ThrAsp Thr Phe Gly Ser Gln 85 90 95 gtc cgg atc aag ggc aag gag acg gaa ttctac ctg tgc atg aac cgc 336 Val Arg Ile Lys Gly Lys Glu Thr Glu Phe TyrLeu Cys Met Asn Arg 100 105 110 aaa ggc aag ctc gtg ggg aag ccc gat ggcacc agc aag gag tgt gtg 384 Lys Gly Lys Leu Val Gly Lys Pro Asp Gly ThrSer Lys Glu Cys Val 115 120 125 ttc atc gag aag gtt ctg gag aac aac tacacg gcc ctg atg tcg gct 432 Phe Ile Glu Lys Val Leu Glu Asn Asn Tyr ThrAla Leu Met Ser Ala 130 135 140 aag tac tcc ggc tgg tac gtg ggc ttc accaag aag ggg cgg ccg cgg 480 Lys Tyr Ser Gly Trp Tyr Val Gly Phe Thr LysLys Gly Arg Pro Arg 145 150 155 160 aag ggc ccc aag acc cgg gag aac cagcag gac gtg cat ttc atg aag 528 Lys Gly Pro Lys Thr Arg Glu Asn Gln GlnAsp Val His Phe Met Lys 165 170 175 cgc tac ccc aag ggg cag ccg gag cttcag aag ccc ttc aag tac acg 576 Arg Tyr Pro Lys Gly Gln Pro Glu Leu GlnLys Pro Phe Lys Tyr Thr 180 185 190 acg gtg acc aag agg tcc cgt cgg atccgg ccc aca cac cct gcc 621 Thr Val Thr Lys Arg Ser Arg Arg Ile Arg ProThr His Pro Ala 195 200 205 taggccaccc cgccgcggcc ctcaggtcgc cctggccacactcacactcc cagaaaactg 681 catcagagga atatttttac atgaaaaata aggattttattgttgacttg aaacccccga 741 tgacaaaaga ctcacgcaaa gggactgtag tcaacccacaggtgcttgtc tctctctagg 801 aacagacaac tctaaactcg tccccagagg aggacttgaatgaggaaacc aacactttga 861 gaaaccaaag tcctttttcc caaaggttct gaaaaaaaaaaaaaaaaaaa ctcgag 917 2 207 PRT Homo sapiens 2 Met Tyr Ser Ala Pro SerAla Cys Thr Cys Leu Cys Leu His Phe Leu 1 5 10 15 Leu Leu Cys Phe GlnVal Gln Val Leu Val Ala Glu Glu Asn Val Asp 20 25 30 Phe Arg Ile His ValGlu Asn Gln Thr Arg Ala Arg Asp Asp Val Ser 35 40 45 Arg Lys Gln Leu ArgLeu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys 50 55 60 His Ile Gln Val LeuGly Arg Arg Ile Ser Ala Arg Gly Glu Asp Gly 65 70 75 80 Asp Lys Tyr AlaGln Leu Leu Val Glu Thr Asp Thr Phe Gly Ser Gln 85 90 95 Val Arg Ile LysGly Lys Glu Thr Glu Phe Tyr Leu Cys Met Asn Arg 100 105 110 Lys Gly LysLeu Val Gly Lys Pro Asp Gly Thr Ser Lys Glu Cys Val 115 120 125 Phe IleGlu Lys Val Leu Glu Asn Asn Tyr Thr Ala Leu Met Ser Ala 130 135 140 LysTyr Ser Gly Trp Tyr Val Gly Phe Thr Lys Lys Gly Arg Pro Arg 145 150 155160 Lys Gly Pro Lys Thr Arg Glu Asn Gln Gln Asp Val His Phe Met Lys 165170 175 Arg Tyr Pro Lys Gly Gln Pro Glu Leu Gln Lys Pro Phe Lys Tyr Thr180 185 190 Thr Val Thr Lys Arg Ser Arg Arg Ile Arg Pro Thr His Pro Ala195 200 205 3 24 DNA Artificial Sequence oligonucleotide primer ZC116763 ggacttgact accgaaggtg tctg 24 4 23 DNA Artificial Sequenceoligonucleotide primer ZC11677 4 gtcgatgtga gccgtaagca gct 23 5 26 DNAArtificial Sequence oligonucleotide primer ZC12053 5 gcatacttgtccccatcctc gccgcg 26 6 621 DNA Artificial Sequence degenerate sequence 6atgtaywsng cnccnwsngc ntgyacntgy ytntgyytnc ayttyytnyt nytntgytty 60cargtncarg tnytngtngc ngargaraay gtngayttym gnathgaygt ngaraarcar 120acnmgngcnm gngaygaygt nwsnmgnaar carytnmgny tntaycaryt ntaywsnmgn 180acnwsnggna arcayathca rgtnytnggn mgnmgnathw sngcnmgngg ngargayggn 240gayaartayg cncarytnyt ngtngaracn gayacnttyg gnwsncargt nmgnathaar 300ggnaargara cngarttyta yytntgyatg aaymgnaarg gnaarytngt nggnaarccn 360gayggnacnw snaargartg ygtnttyath garaargtny tngaraayaa ytayacngcn 420ytnatgwsng cnaartayws nggntggtay gtnggnttya cnaaraargg nmgnccnmgn 480aarggnccna aracnmgnga raaycarcar gaygtncayt tyatgaarmg ntayccnaar 540ggncarccng arytncaraa rccnttyaar tayacnacng tnacnaarmg nwsnmgnmgn 600athmgnccna cncayccngc n 621 7 47 DNA Artificial Sequence oligonucleotideprimer ZC12652 7 tatttatcta gactggttcc gcgtgccgcc gaggagaacg tggactt 478 33 DNA Artificial Sequence oligonucleotide primer ZC12631 8 gtatttgtcgactcaggcag ggtgtgtggg ccg 33 9 22 DNA Artificial Sequenceoligonucleotide primer ZC15290 9 gccgaggaga acgtggactt cc 22 10 47 DNAArtificial Sequence oligonucleotide primer ZC15270 10 tatttatctagagatgacga tgacaaggcc gaggagaacg tggactt 47 11 41 DNA ArtificialSequence oligonucleotide primer ZC13497 11 agcattgcta aagaagaaggtgtaagcttg gacaagagag a 41 12 63 DNA Artificial Sequence oligonucleotideprimer ZC15131 12 ggtgtaagct tggacaagag agaggagaac gtggacttcc gcatccacgtggagaaccag 60 acg 63 13 39 DNA Artificial Sequence oligonucleotideprimer ZC15134 13 ccggctgtag agctggtaca gccgcagctg cttacggct 39 14 42DNA Artificial Sequence oligonucleotide primer ZC13529 14 cttcagaagcccttcaagta cacgacggtg accaagaggt cc 42 15 61 DNA Artificial Sequenceoligonucleotide primer ZC13525 15 acgacggtga ccaagaggtc ccgtcggatccggcccacac accctgccta gggggaattc 60 g 61 16 61 DNA Artificial Sequenceoligonucleotide primer ZC13526 16 caaacaggca gccctagaat actagtgtcgactcgaggat ccgaattccc cctaggcagg 60 g 61 17 44 DNA Artificial Sequenceoligonucleotide primer ZC13528 17 ctcaaaaatt ataaaaatat ccaaacaggcagccctagaa tact 44 18 186 DNA Artificial Sequence oligonucleotide primerZC15132 18 gtaccgcgag cagttcccgt caatccctcc ccccttacac aggatgtccatattaggaca 60 tctgcgtctc gaggccaccg tggttgagcc cgacactcat tcataaaacgcttgttataa 120 aagcagtggc tgcggcgcct cgtactccaa ccgcatctgc agcgagcaactgagaagcca 180 aggatc 186 19 141 DNA Artificial Sequence 5′ linkersequence 19 agcattgctg ctaaagaaga aggtgtaagc ttggacaaga gagaggagaacgtggacttc 60 cgcatccacg tggagaacca gacgcgggct cgggacgatg tgagccgtaagcagctgcgg 120 ctgtaccagc tctacagccg g 141 20 144 DNA ArtificialSequence 3′ linker sequence 20 cttcagaagc ccttcaagta cacgacggtgaccaagaggt cccgtcggat ccggcccaca 60 caccctgcct agggggaatt cggatcctcgagtcgacact agtattctag ggctgcctgt 120 ttggatattt ttataatttt tgag 144 21243 PRT Homo sapiens 21 Met Ala Ala Ala Ile Ala Ser Ser Leu Ile Arg GlnLys Arg Gln Ala 1 5 10 15 Arg Glu Ser Asn Ser Asp Arg Val Ser Ala SerLys Arg Arg Ser Ser 20 25 30 Pro Ser Lys Asp Gly Arg Ser Leu Cys Glu ArgHis Val Leu Gly Val 35 40 45 Phe Ser Lys Val Arg Phe Cys Ser Gly Arg LysArg Pro Val Arg Arg 50 55 60 Arg Pro Glu Pro Gln Leu Lys Gly Ile Val ThrArg Leu Phe Ser Gln 65 70 75 80 Gln Gly Tyr Phe Leu Gln Met His Pro AspGly Thr Ile Asp Gly Thr 85 90 95 Lys Asp Glu Asn Ser Asp Tyr Thr Leu PheAsn Leu Ile Pro Val Gly 100 105 110 Leu Arg Val Val Ala Ile Gln Gly ValLys Ala Ser Leu Tyr Val Ala 115 120 125 Met Asn Gly Glu Gly Tyr Leu TyrSer Ser Asp Val Phe Thr Pro Glu 130 135 140 Cys Lys Phe Lys Glu Ser ValPhe Glu Asn Tyr Tyr Val Ile Tyr Ser 145 150 155 160 Ser Thr Leu Tyr ArgGln Gln Glu Ser Gly Arg Ala Trp Phe Leu Gly 165 170 175 Leu Asn Lys GluGly Gln Ile Met Lys Gly Asn Arg Val Lys Lys Thr 180 185 190 Lys Pro SerSer His Phe Val Pro Lys Pro Ile Glu Val Cys Met Tyr 195 200 205 Arg GluPro Ser Leu His Glu Ile Gly Glu Lys Gln Gly Arg Ser Arg 210 215 220 LysSer Ser Gly Thr Pro Thr Met Asn Gly Gly Lys Val Val Asn Gln 225 230 235240 Asp Ser Thr 22 168 PRT Homo sapiens 22 Met Ala Ser Lys Glu Pro GlnLeu Lys Gly Ile Val Thr Arg Leu Phe 1 5 10 15 Ser Gln Gln Gly Tyr PheLeu Gln Met His Pro Asp Gly Thr Ile Asp 20 25 30 Gly Thr Lys Asp Glu AsnSer Asp Tyr Thr Leu Phe Asn Leu Ile Pro 35 40 45 Val Gly Leu Arg Val ValAla Ile Gln Gly Val Lys Ala Ser Leu Tyr 50 55 60 Val Ala Met Asn Gly GluGly Tyr Leu Tyr Ser Ser Asp Val Phe Thr 65 70 75 80 Pro Glu Cys Lys PheLys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile 85 90 95 Tyr Ser Ser Thr LeuTyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe 100 105 110 Leu Gly Leu AsnLys Glu Gly Gln Ile Met Lys Gly Asn Arg Val Glu 115 120 125 Lys Thr LysPro Ser Ser His Phe Val Pro Lys Pro Ile Glu Val Cys 130 135 140 Met TyrArg Glu Pro Ser Leu His Glu Ile Gly Glu Asn Lys Gly Val 145 150 155 160Gln Gly Lys Phe Trp Thr Pro Pro 165 23 247 PRT Homo sapiens 23 Met AlaAla Ala Ile Ala Ser Gly Leu Ile Arg Gln Lys Arg Gln Ala 1 5 10 15 ArgGlu Gln His Trp Asp Arg Pro Ser Ala Ser Arg Arg Arg Ser Ser 20 25 30 ProSer Lys Asn Arg Gly Leu Cys Asn Gly Asn Leu Val Asp Ile Phe 35 40 45 SerLys Val Arg Ile Phe Gly Leu Lys Lys Arg Arg Leu Arg Arg Gln 50 55 60 AspPro Gln Leu Lys Gly Ile Val Thr Arg Leu Tyr Cys Arg Gln Gly 65 70 75 80Tyr Tyr Leu Gln Met His Pro Asp Gly Ala Leu Asp Gly Thr Lys Asp 85 90 95Asp Ser Thr Asn Ser Thr Leu Phe Asn Leu Ile Pro Val Gly Leu Arg 100 105110 Val Val Ala Ile Gln Gly Val Lys Thr Gly Leu Tyr Ile Ala Met Asn 115120 125 Gly Glu Gly Tyr Leu Tyr Pro Ser Glu Leu Phe Thr Pro Glu Cys Lys130 135 140 Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser SerMet 145 150 155 160 Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe LeuGly Leu Asn 165 170 175 Lys Glu Gly Gln Ala Met Lys Gly Asn Arg Val LysLys Thr Lys Pro 180 185 190 Ala Ala His Phe Leu Pro Lys Pro Leu Glu ValAla Met Tyr Arg Glu 195 200 205 Pro Ser Leu His Asp Val Gly Glu Thr ValPro Lys Pro Gly Val Thr 210 215 220 Pro Ser Lys Ser Thr Ser Ala Ser AlaIle Met Asn Gly Gly Lys Pro 225 230 235 240 Val Asn Lys Ser Lys Thr Thr245 24 245 PRT Homo sapiens 24 Met Ala Ala Ala Ile Ala Ser Ser Leu IleArg Gln Lys Arg Gln Ala 1 5 10 15 Arg Glu Arg Glu Lys Ser Asn Ala CysLys Cys Val Ser Ser Pro Ser 20 25 30 Lys Gly Lys Thr Ser Cys Asp Lys AsnLys Leu Asn Val Phe Ser Arg 35 40 45 Val Lys Leu Phe Gly Ser Lys Lys ArgArg Arg Arg Arg Pro Glu Pro 50 55 60 Gln Leu Lys Gly Ile Val Thr Lys LeuTyr Ser Arg Gln Gly Tyr His 65 70 75 80 Leu Gln Leu Gln Ala Asp Gly ThrIle Asp Gly Thr Lys Asp Glu Asp 85 90 95 Ser Thr Tyr Thr Leu Phe Asn LeuIle Pro Val Gly Leu Arg Val Val 100 105 110 Ala Ile Gln Gly Val Gln ThrLys Leu Tyr Leu Ala Met Asn Ser Glu 115 120 125 Gly Tyr Leu Tyr Thr SerGlu Leu Phe Thr Pro Glu Cys Lys Phe Lys 130 135 140 Glu Ser Val Phe GluAsn Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr 145 150 155 160 Arg Gln GlnGln Ser Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu 165 170 175 Gly GluIle Met Lys Gly Asn His Val Lys Lys Asn Lys Pro Ala Ala 180 185 190 HisPhe Leu Pro Lys Pro Leu Lys Val Ala Met Tyr Lys Glu Pro Ser 195 200 205Leu His Asp Leu Thr Glu Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr 210 215220 Lys Ser Arg Ser Val Ser Gly Val Leu Asn Gly Gly Lys Ser Met Ser 225230 235 240 His Asn Glu Ser Thr 245 25 225 PRT Homo sapiens 25 Met AlaAla Leu Ala Ser Ser Leu Ile Arg Gln Lys Arg Glu Val Arg 1 5 10 15 GluPro Gly Gly Ser Arg Pro Val Ser Ala Gln Arg Arg Val Cys Pro 20 25 30 ArgGly Thr Lys Ser Leu Cys Gln Lys Gln Leu Leu Ile Leu Leu Ser 35 40 45 LysVal Arg Leu Cys Gly Gly Arg Pro Ala Arg Pro Asp Arg Gly Pro 50 55 60 GluPro Gln Leu Lys Gly Ile Val Thr Lys Leu Phe Cys Arg Gln Gly 65 70 75 80Phe Tyr Leu Gln Ala Asn Pro Asp Gly Ser Ile Gln Gly Thr Pro Glu 85 90 95Asp Thr Ser Ser Phe Thr His Phe Asn Leu Ile Pro Val Gly Leu Arg 100 105110 Val Val Thr Ile Gln Ser Ala Lys Leu Gly His Tyr Met Ala Met Asn 115120 125 Ala Glu Gly Leu Leu Tyr Ser Ser Pro His Phe Thr Ala Glu Cys Arg130 135 140 Phe Lys Glu Cys Val Phe Glu Asn Tyr Tyr Val Leu Tyr Ala SerAla 145 150 155 160 Leu Tyr Arg Gln Arg Arg Ser Gly Arg Ala Trp Tyr LeuGly Leu Asp 165 170 175 Lys Glu Gly Gln Val Met Lys Gly Asn Arg Val LysLys Thr Lys Ala 180 185 190 Ala Ala His Phe Leu Pro Lys Leu Leu Glu ValAla Met Tyr Gln Glu 195 200 205 Pro Ser Leu His Ser Val Pro Glu Ala SerPro Ser Ser Pro Pro Ala 210 215 220 Pro 225 26 206 PRT Homo sapiens 26Met Ser Gly Pro Gly Thr Ala Ala Val Ala Leu Leu Pro Ala Val Leu 1 5 1015 Leu Ala Leu Leu Ala Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala Pro 20 2530 Thr Ala Pro Asn Gly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu 35 4045 Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gln Pro 50 5560 Lys Glu Ala Ala Val Gln Ser Gly Ala Gly Asp Tyr Leu Leu Gly Ile 65 7075 80 Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe His Leu 8590 95 Gln Ala Leu Pro Asp Gly Arg Ile Gly Gly Ala His Ala Asp Thr Arg100 105 110 Asp Ser Leu Leu Glu Leu Ser Pro Val Glu Arg Gly Val Val SerIle 115 120 125 Phe Gly Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser LysGly Lys 130 135 140 Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr PheLys Glu Ile 145 150 155 160 Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu SerTyr Lys Tyr Pro Gly 165 170 175 Met Phe Ile Ala Leu Ser Lys Asn Gly LysThr Lys Lys Gly Asn Arg 180 185 190 Val Ser Pro Thr Met Lys Val Thr HisPhe Leu Pro Arg Leu 195 200 205 27 208 PRT Homo sapiens 27 Met Ala LeuGly Gln Lys Leu Phe Ile Thr Met Ser Arg Gly Ala Gly 1 5 10 15 Arg LeuGln Gly Thr Leu Trp Ala Leu Val Phe Leu Gly Ile Leu Val 20 25 30 Gly MetVal Val Pro Ser Pro Ala Gly Thr Arg Ala Asn Asn Thr Leu 35 40 45 Leu AspSer Arg Gly Trp Gly Thr Leu Leu Ser Arg Ser Arg Ala Gly 50 55 60 Leu AlaGly Glu Ile Ala Gly Val Asn Trp Glu Ser Gly Tyr Leu Val 65 70 75 80 GlyIle Lys Arg Gln Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe 85 90 95 HisLeu Gln Val Leu Pro Asp Gly Arg Ile Ser Gly Thr His Glu Glu 100 105 110Asn Pro Tyr Ser Leu Leu Glu Ile Ser Thr Val Glu Arg Gly Val Val 115 120125 Ser Leu Phe Gly Val Arg Ser Ala Leu Phe Val Ala Met Asn Ser Lys 130135 140 Gly Arg Leu Tyr Ala Thr Pro Ser Phe Gln Glu Glu Cys Lys Phe Arg145 150 155 160 Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser AspLeu Tyr 165 170 175 Gln Gly Thr Tyr Ile Ala Leu Ser Lys Tyr Gly Arg ValLys Arg Gly 180 185 190 Ser Lys Val Ser Pro Ile Met Thr Val Thr His PheLeu Pro Arg Ile 195 200 205 28 155 PRT Homo sapiens 28 Met Ala Ala GlySer Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 Gly Ser GlyAla Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys LysAsn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 Val Asp GlyVal Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 Gln Ala GluGlu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80 Arg TyrLeu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95 Val ThrAsp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110 AsnThr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys 130 135140 Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155 29 155 PRTHomo sapiens 29 Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr GluLys Phe 1 5 10 15 Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu LeuTyr Cys Ser 20 25 30 Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly ThrVal Asp Gly 35 40 45 Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln LeuSer Ala Glu 50 55 60 Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr GlyGln Tyr Leu 65 70 75 80 Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser GlnThr Pro Asn Glu 85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn HisTyr Asn Thr Tyr 100 105 110 Ile Ser Lys Lys His Ala Glu Lys Asn Trp PheVal Gly Leu Lys Lys 115 120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg ThrHis Tyr Gly Gln Lys Ala 130 135 140 Ile Leu Phe Leu Pro Leu Pro Val SerSer Asp 145 150 155 30 208 PRT Homo sapiens 30 Met Trp Lys Trp Ile LeuThr His Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15 Pro Gly Cys Cys CysCys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 Val Pro Val Thr CysGln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 Ala Thr Asn Ser SerSer Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly 50 55 60 Arg His Val Arg SerTyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 Lys Leu Phe SerPhe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly 85 90 95 Lys Val Ser GlyThr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu 100 105 110 Ile Thr SerVal Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser 115 120 125 Asn TyrTyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 GluPhe Asn Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly 145 150 155160 Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met 165170 175 Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr180 185 190 Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val HisSer 195 200 205 31 194 PRT Homo sapiens 31 Met His Lys Trp Ile Leu ThrTrp Ile Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15 Ser Cys Phe His Ile IleCys Leu Val Gly Thr Ile Ser Leu Ala Cys 20 25 30 Asn Asp Met Thr Pro GluGln Met Ala Thr Asn Val Asn Cys Ser Ser 35 40 45 Pro Glu Arg His Thr ArgSer Tyr Asp Tyr Met Glu Gly Gly Asp Ile 50 55 60 Arg Val Arg Arg Leu PheCys Arg Thr Gln Trp Tyr Leu Arg Ile Asp 65 70 75 80 Lys Arg Gly Lys ValLys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn 85 90 95 Ile Met Glu Ile ArgThr Val Ala Val Gly Ile Val Ala Ile Lys Gly 100 105 110 Val Glu Ser GluPhe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115 120 125 Ala Lys LysGlu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu 130 135 140 Glu AsnHis Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly 145 150 155 160Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly 165 170175 Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala 180185 190 Ile Thr 32 233 PRT Homo sapiens 32 Met Gly Ser Pro Arg Ser AlaLeu Ser Cys Leu Leu Leu His Leu Leu 1 5 10 15 Val Leu Cys Leu Gln AlaGln Glu Gly Pro Gly Arg Gly Pro Ala Leu 20 25 30 Gly Arg Glu Leu Ala SerLeu Phe Arg Ala Gly Arg Glu Pro Gln Gly 35 40 45 Val Ser Gln Gln His ValArg Glu Gln Ser Leu Val Thr Asp Gln Leu 50 55 60 Ser Arg Arg Leu Ile ArgThr Tyr Gln Leu Tyr Ser Arg Thr Ser Gly 65 70 75 80 Lys His Val Gln ValLeu Ala Asn Lys Arg Ile Asn Ala Met Ala Glu 85 90 95 Asp Gly Asp Pro PheAla Lys Leu Ile Val Glu Thr Asp Thr Phe Gly 100 105 110 Ser Arg Val ArgVal Arg Gly Ala Glu Thr Gly Leu Tyr Ile Cys Met 115 120 125 Asn Lys LysGly Lys Leu Ile Ala Lys Ser Asn Gly Lys Gly Lys Asp 130 135 140 Cys ValPhe Thr Glu Ile Val Leu Glu Asn Asn Tyr Thr Ala Leu Gln 145 150 155 160Asn Ala Lys Tyr Glu Gly Trp Tyr Met Ala Phe Thr Arg Lys Gly Arg 165 170175 Pro Arg Lys Gly Ser Lys Thr Arg Gln His Gln Arg Glu Val His Phe 180185 190 Met Lys Arg Leu Pro Arg Gly His His Thr Thr Glu Gln Ser Leu Arg195 200 205 Phe Glu Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg GlySer 210 215 220 Gln Arg Thr Trp Ala Pro Glu Pro Arg 225 230 33 268 PRTHomo sapiens 33 Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His LeuIle Leu 1 5 10 15 Ser Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro LysGly Gln Pro 20 25 30 Gly Pro Ala Ala Thr Asp Arg Asn Pro Ile Gly Ser SerSer Arg Gln 35 40 45 Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser SerSer Pro Ala 50 55 60 Ala Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln SerSer Phe Gln 65 70 75 80 Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu TyrCys Arg Val Gly 85 90 95 Ile Gly Phe His Leu Gln Ile Tyr Pro Asp Gly LysVal Asn Gly Ser 100 105 110 His Glu Ala Asn Met Leu Ser Val Leu Glu IlePhe Ala Val Ser Gln 115 120 125 Gly Ile Val Gly Ile Arg Gly Val Phe SerAsn Lys Phe Leu Ala Met 130 135 140 Ser Lys Lys Gly Lys Leu His Ala SerAla Lys Phe Thr Asp Asp Cys 145 150 155 160 Lys Phe Arg Glu Arg Phe GlnGlu Asn Ser Tyr Asn Thr Tyr Ala Ser 165 170 175 Ala Ile His Arg Thr GluLys Thr Gly Arg Glu Trp Tyr Val Ala Leu 180 185 190 Asn Lys Arg Gly LysAla Lys Arg Gly Cys Ser Pro Arg Val Lys Pro 195 200 205 Gln His Ile SerThr His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln 210 215 220 Pro Glu LeuSer Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro 225 230 235 240 SerPro Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr 245 250 255Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265 34 208 PRT Homosapiens 34 Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln AspAla 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser ProVal Leu 20 25 30 Leu Ser Asp His Leu Gly Gln Ser Glu Ala Gly Gly Leu ProArg Gly 35 40 45 Pro Ala Val Thr Asp Leu Asp His Leu Lys Gly Ile Leu ArgArg Arg 50 55 60 Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe ProAsn Gly 65 70 75 80 Thr Ile Gln Gly Thr Arg Lys Asp His Ser Arg Phe GlyIle Leu Glu 85 90 95 Phe Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg GlyVal Asp Ser 100 105 110 Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu LeuTyr Gly Ser Glu 115 120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu GlnPhe Glu Glu Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr LysHis Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu Asn LysAsp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gln LysPhe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro Asp Lys Val ProGlu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205 35 239 PRT Homosapiens 35 Met Gly Leu Ile Trp Leu Leu Leu Leu Ser Leu Leu Glu Pro GlyTrp 1 5 10 15 Pro Ala Ala Gly Pro Gly Ala Arg Leu Arg Arg Asp Ala GlyGly Arg 20 25 30 Gly Gly Val Tyr Glu His Leu Gly Gly Ala Pro Arg Arg ArgLys Leu 35 40 45 Tyr Cys Ala Thr Lys Tyr His Leu Gln Leu His Pro Ser GlyArg Val 50 55 60 Asn Gly Ser Leu Glu Asn Ser Ala Tyr Ser Ile Leu Glu IleThr Ala 65 70 75 80 Val Glu Val Gly Ile Val Ala Ile Arg Gly Leu Phe SerGly Arg Tyr 85 90 95 Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser GluHis Tyr Ser 100 105 110 Ala Glu Cys Glu Phe Val Glu Arg Ile His Glu LeuGly Tyr Asn Thr 115 120 125 Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser SerThr Pro Gly Ala Arg 130 135 140 Arg Gln Pro Ser Ala Glu Arg Leu Trp TyrVal Ser Val Asn Gly Lys 145 150 155 160 Gly Arg Pro Arg Arg Gly Phe LysThr Arg Arg Thr Gln Lys Ser Ser 165 170 175 Leu Phe Leu Pro Arg Val LeuAsp His Arg Asp His Glu Met Val Arg 180 185 190 Gln Leu Gln Ser Gly LeuPro Arg Pro Pro Gly Lys Gly Val Gln Pro 195 200 205 Arg Arg Arg Arg GlnLys Gln Ser Pro Asp Asn Leu Glu Pro Ser His 210 215 220 Val Gln Ala SerArg Leu Gly Ser Gln Leu Glu Ala Ser Ala His 225 230 235 36 11 PRTArtificial Sequence FGF family motif 36 Cys Xaa Phe Xaa Glu Glu Glu GluGlu Glu Tyr 1 5 10 37 4 PRT Artificial Sequence dibasic cleavage peptide37 Arg Xaa Xaa Arg 1 38 1023 DNA Mus musculus CDS (1)...(624) 38 atg tattca gcg ccc tcc gcc tgc act tgc ctg tgt tta cac ttt cta 48 Met Tyr SerAla Pro Ser Ala Cys Thr Cys Leu Cys Leu His Phe Leu 1 5 10 15 ctg ctgtgc ttc cag gtt cag gtg ttg gca gcc gag gag aat gtg gac 96 Leu Leu CysPhe Gln Val Gln Val Leu Ala Ala Glu Glu Asn Val Asp 20 25 30 ttc cgc atccac gtg gag aac cag acg cgg gct cga gat gat gtg agt 144 Phe Arg Ile HisVal Glu Asn Gln Thr Arg Ala Arg Asp Asp Val Ser 35 40 45 cgg aag cag ctgcgc ttg tac cag ctc tat agc agg acc agt ggg aag 192 Arg Lys Gln Leu ArgLeu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys 50 55 60 cac att caa gtc ctgggc cgt agg atc agt gcc cgt ggc gag gac ggg 240 His Ile Gln Val Leu GlyArg Arg Ile Ser Ala Arg Gly Glu Asp Gly 65 70 75 80 gac aag tat gcc cagctc cta gtg gag aca gat acc ttc ggg agt caa 288 Asp Lys Tyr Ala Gln LeuLeu Val Glu Thr Asp Thr Phe Gly Ser Gln 85 90 95 gtc cgg atc aag ggc aaggag aca gaa ttc tac ctg tgt atg aac cga 336 Val Arg Ile Lys Gly Lys GluThr Glu Phe Tyr Leu Cys Met Asn Arg 100 105 110 aaa ggc aag ctc gtg gggaag cct gat ggt act agc aag gag tgc gtg 384 Lys Gly Lys Leu Val Gly LysPro Asp Gly Thr Ser Lys Glu Cys Val 115 120 125 ttc att gag aag gtt ctggaa aac aac tac acg gcc ctg atg tct gcc 432 Phe Ile Glu Lys Val Leu GluAsn Asn Tyr Thr Ala Leu Met Ser Ala 130 135 140 aag tac tct ggt tgg tatgtg ggc ttc acc aag aag ggg cgg cct cgc 480 Lys Tyr Ser Gly Trp Tyr ValGly Phe Thr Lys Lys Gly Arg Pro Arg 145 150 155 160 aag ggt ccc aag acccgc gag aac cag caa gat gta cac ttc atg aag 528 Lys Gly Pro Lys Thr ArgGlu Asn Gln Gln Asp Val His Phe Met Lys 165 170 175 cgt tac ccc aag ggacag gcc gag ctg cag aag ccc ttc aaa tac acc 576 Arg Tyr Pro Lys Gly GlnAla Glu Leu Gln Lys Pro Phe Lys Tyr Thr 180 185 190 aca gtc acc aag cgatcc cgg cgg atc cgc ccc act cac ccc ggc tag 624 Thr Val Thr Lys Arg SerArg Arg Ile Arg Pro Thr His Pro Gly * 195 200 205 gtccggccac actcacccccccagagaact acatcagagg aatattttta catgaaaaat 684 aaggaagaat ctctatttttgtacattgtg tttaaaagaa gacaaaaact gaacctaaag 744 tcttgggagg aggggcgataggattccact gttgacctga accccatgac aaaggactca 804 cacaagggga ccgctgtcaacccacaggtg cttgcctctc tctaggaggt gacaattcaa 864 aactcatccc cagaggaggacttgaacgag gaaactgcga gaaaccaaag tcctttcccc 924 ccaaaggttc tgaaagcaaacaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 984 aaaaaaaaaa aaaaaaaaaagggcggccgc tctagagga 1023 39 207 PRT Mus musculus 39 Met Tyr Ser Ala ProSer Ala Cys Thr Cys Leu Cys Leu His Phe Leu 1 5 10 15 Leu Leu Cys PheGln Val Gln Val Leu Ala Ala Glu Glu Asn Val Asp 20 25 30 Phe Arg Ile HisVal Glu Asn Gln Thr Arg Ala Arg Asp Asp Val Ser 35 40 45 Arg Lys Gln LeuArg Leu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys 50 55 60 His Ile Gln ValLeu Gly Arg Arg Ile Ser Ala Arg Gly Glu Asp Gly 65 70 75 80 Asp Lys TyrAla Gln Leu Leu Val Glu Thr Asp Thr Phe Gly Ser Gln 85 90 95 Val Arg IleLys Gly Lys Glu Thr Glu Phe Tyr Leu Cys Met Asn Arg 100 105 110 Lys GlyLys Leu Val Gly Lys Pro Asp Gly Thr Ser Lys Glu Cys Val 115 120 125 PheIle Glu Lys Val Leu Glu Asn Asn Tyr Thr Ala Leu Met Ser Ala 130 135 140Lys Tyr Ser Gly Trp Tyr Val Gly Phe Thr Lys Lys Gly Arg Pro Arg 145 150155 160 Lys Gly Pro Lys Thr Arg Glu Asn Gln Gln Asp Val His Phe Met Lys165 170 175 Arg Tyr Pro Lys Gly Gln Ala Glu Leu Gln Lys Pro Phe Lys TyrThr 180 185 190 Thr Val Thr Lys Arg Ser Arg Arg Ile Arg Pro Thr His ProGly 195 200 205 40 21 DNA Artificial Sequence Oligonucleotide primerZC17579 40 aaaggcaagc tcgtggggaa g 21 41 22 DNA Artificial SequenceOligonucleotide primer ZC17578 41 tcgcttggtg actgtggtgt at 22 42 19 DNAArtificial Sequence Oligonucleotide primer ZC19567 42 atgtattcagcgccctccg 19 43 19 DNA Artificial Sequence Oligonucleotide primerZC19633 43 cgagcccgcg tctggttct 19

We claim:
 1. A method of reducing infarct volume in a mammal diagnosedas having a cerebrovascular ischemic stroke comprising: (1) determininginfarct volume in the mammal; (2) administering a pharmaceuticalcomposition comprising a polypeptide that is at least 80% identical toan amino acid sequence as shown in SEQ ID NO: 2 from amino acid residue28 (Glu) to amino acid residue 175 (Met) sufficient to reduce infarctvolume; (3) determining infarct volume in the mammal; and (4) comparingthe infarct volume of step 1 to step
 4. 2. The method of claim 1 whereinthe pharmaceutical composition is a polypeptide that is at least 80%identical to the amino acid sequence as shown in SEQ ID NO: 2 fromresidue 28 (Glu) to 196 (Lys).
 3. The method of claim 1 wherein thepharmaceutical composition is a polypeptide is the amino acid sequenceas shown in SEQ ID NO: 2 from residue 28 (Glu) to residue 196 (Lys). 4.A method for treating a patient who has suffered an injury to thecentral nervous system, comprising administering to the patient apharmaceutical composition comprising a fibroblast growth factor (FGF)homolog polypeptide, wherein said polypeptide comprises a sequence ofamino acids that is at least 80% identical to the amino acid sequence asshown in SEQ ID NO: 2 from amino acid residue 28 (Glu) to 175 (Met), inamount sufficient to improve functional recovery in the patient.
 5. Themethod of claim 4, wherein the injury to the central nervous system isan ischemic event.
 6. The method of claim 4, wherein the ischemic eventis a stroke.
 7. The method of claim 4, wherein improved functionalrecovery is defined as a patient score of at least 3 on a Rankin strokescale.
 8. The method of claim 4, wherein improved functional recovery isdefined as a patient score of lower than 75 on a Barthel's scale.
 9. Themethod of claim 4, wherein said polypeptide encoded by saidpolynucleotide is at least 90% identical to the amino acid sequence asshown in SEQ ID NO: 2 from amino acid residue 28 (Glu) to residue 196(Lys).
 10. The method of claim 4, wherein said polypeptide encoded bysaid polynucleotide is at least 80% identical to the amino acid sequenceas shown in SEQ ID NO: 2 from amino acid residue 28 (Glu) to residue 207(Ala).
 11. A method of increasing cartilage deposition in a patientcomprising: administering a pharmaceutical composition comprising afibroblast growth factor (FGF) homolog polypeptide to a fibrocartilage,hyaline, or elastic cartilage injury in said patient, wherein saidpolypeptide comprises a sequence of amino acids that is at least 80%identical to the amino acid sequence as shown in SEQ ID NO: 2 from aminoacid residue 28 (Glu) to 175 (Met).
 12. The method of claim 11, whereinthe administration of the composition is selected from the groupconsisting of intracartilaginous administration; intraartictilaradministration; intravenous; intramuscular; and topical administration.